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qPCR BioStatistics
& BioInformatics
P001 A
Rapid Bioinformatic Engine for Multiplexed qPCR Design Biosearch
Technologies, Inc., Multiplexed
qPCR remains a
challenging endeavor for reasons that include: 1) designing assays to
combine
without interference, 2) resolving fluorophores using the optics of
each
real-time instrument, and 3) optimizing and validating each assay's
performance. Here, we address each of these issues when developing
several
pentaplexed assays that target genes from human and mouse. Each assay
was
designed using a free, online, software program that carefully
considers
inter-oligo interactions while simultaneously building its multiplexed
set.
Situations of disproportionate copy number present a particular
challenge upon
multiplexed performance; additional validation is needed to define the
limits
of a multiplexed set, as compared to individually amplified assays. P002 Unit of
Medical Statistics and
Biometry, Fondazione IRCCS Istituto Nazionale Tumori, There are two
major approaches of
real-time PCR quantification: the absolute and the relative method. The
latter
evaluates the change in expression of the target gene relative to a
reference
gene, whereas the absolute method uses a standard curve to quantify
unknown
amount of nucleic acids in a target sample. From a statistical view
point, the
standard curve corresponds to the simple linear regression model and
thus the
absolute quantification method allows to exploit the methodological
background
of the linear regression theory. By means of a technique known as
inverse
regression, the fitted standard curve is used as calibrator to estimate
the
unknown nucleic acid concentration in the target sample. Several
approaches
have been proposed for constructing confidence intervals in inverse
regression.
We propose an user-friendly algorithm, named BCI (Bootstrap Confidence
Interval), specifically designed to compute bootstrap-t confidence
interval for
nucleic acid concentration by absolute real-time PCR. The algorithm has
been
written in R language an open-source statistical software and provides
the
bootstrap estimate of the unknown concentration both in logarithmic
scale and
in its original scale as copy number together with the lower and upper
limits
of the 100(1-α)% bootstrap-t confidence interval of the unknown
concentration.
Users can modify the number of bootstrap resampling and the confidence
level
(1- α) of the bootstrap-t confidence interval. P003 Data
analysis for gene quantification and expression profiling using GenEx. MultiD
Analyses AB, As the data
size and complexity from
qPCR projects increase, the need for comprehensive automated or
semi-automated
software tools increase rapidly. Software tools can provide support
after data
collection by providing data pre-processing, statistical analysis and
visualization capabilities, often for hypothesis generating purposes.
Alternatively they can be used prior to experimental realization by
helping to
define experimental design parameters for hypothesis validation assays.
The
GenEx software from MultiD Analyses AB provides all of the capabilities
mentioned above. Performing accurate qPCR data pre-processing is very
important, particularly for quantification purposes. Many steps are
usually
implemented and it is useful to follow protocols in order to avoid
introduction
of unwarranted processing variability and bias. The protocol available
in GenEx
is easy to adapt to user-specific needs while at the same time
comprehensive
enough to enable users to easily perform accurate pre-processing. As
scientists
we believe observations we make are manifestations of rational
processes. Our
challenge is to identify the particular rational process that we want
to study
while minimizing contributions from rational processes that would
obscure the
understanding of our particular study. Contributions from unwanted
processes
are often called random although the underlying processes may not be.
Tools to
differentiate between contributions from desired and unwanted processes
include
parametric and non-parametric statistical tests, scatterplots,
principle
component analyses and neural network analyses. These tests and
analyses and more
are available in GenEx. Validation of scientific conclusions is not
absolute,
but based on reproducibility. No scientific theory is above scrutiny
and
potential revision. However, based on certain assumptions, an
increasing number
of observations that supports a particular conclusion will also
increase our
confidence that the particular conclusion is going to continue to be
supported
by future observations. We may thus define a level of confidence by
which we
would assign our conclusion to be “true”. A good experimental design
aimed to
validate a hypothesis should therefore include the number of necessary
observations needed to obtain the desired level of confidence, before
realization of the experiment. Based on a hypothesis of an observed
effect,
including desired level of confidence, amplitude of desired observed
effect and
variability of confounding effects, GenEx can calculate the number of
necessary
observations. The presentation will focus on important considerations
for
running qPCR experiments and ways the GenEx software may provide
support. P004 An
alternative way for Real Time PCR data Analysis Instituto
Portugues Oncologia de
Lisboa Francisco Gentil, EPE, Virology INTRODUCTION
- Real Time PCR is a
methodology with increasing applications in the clinical laboratory.
This new
and revolutionary method combines the PCR chemistry with the
fluorescent
probe/dye detection of the amplified product, all in the same reaction
tube.
Since the equipment used can record the emission of fluorescence during
all the
cycles of amplification, a significant increase of the PCR product is
directly
linked with the initial amount of target DNA. In Real Time PCR, we can
determine a fixed fluorescent treshold, above the background. When the
PCR
product that we want to detect cross this threshold, we can determine a
parameter named Cycle Threshold (Ct). All the equipment used in Real
Time PCR
experiments have some kind of software to analyse the data, namely the
analysis
of the expression of Ct value relatively to the log[DNA]. However, this
software doesn’t give much details regarding the linear regression: it
only
calculates the slope, y intercept and coefficient of determination
(R2). AIM - Development
of an Excel sheet that calculates several parameters regarding the
linear
regression and assay validation/calibration. RESULTS - We have
developed an
Excel sheet that uses the data from calibration data (4 different
concentrations of Virus) in order to determine the following parameters: • Slope • Y Intercept • Coefficient
of Determination • Efficiency
Amplification • Detection
and quantification limit
(analytical and method) • Standard
Error (RMSE) • P-Value
associated with the linear
regression (validation) Relatively to
the linear regression
parameters, we have introduced the ANOVA (Analysis of Variance) in
order to
determine the Sum of Squares of: Regression, Residual, Lack of Fit and
Pure
Error. With this approach we can make an objective analyses of the
goodness of
fit, residuals, determine outliers and the confidence interval of the
samples
with viral load. Since our laboratory is accredited (ISO 17025: 2005),
it also
helps in the maintenance of records relatively to batch/expire date of
DNA/RNA
extraction kit, primers, probes, master mix, internal control
(amplification)
and the reaction plates. CONCLUSION - This Excel sheet is a very good
alternative to the data analysis of the standard software present in
the
various Real Time PCR equipments. P005 RefGenes:
new tool to find suitable reference genes 1ETH
Zurich & NEBION AG, Reference
genes (or “housekeeping
genes”) are often used as internal controls for transcript
quantification
assays. Often, classical reference genes such as GAPDH or TUBB are not
suitable
for one’s own experimental condition because their expression varies
significantly. The goal of RefGenes is to identify, from a genome-wide
set of
genes, those that are most suitable for a given condition. This is
achieved by
screening Genevestigator’s large expression compendium (>27,000
microarrays). Validation experiments on plant and animal qRT-PCR
experiments
showed that genes found through RefGenes performed significantly better
as
normalizers than classical reference genes. P006 A
High Throughput, Quantitative Real Time PCR Method for the
Determination of
Copy Number Variation in Knockout Mice 1Applied
Biosystems, Warrington, United Kingdom; 2Wellcome Trust
Sanger
Institute, Cambridge, United Kingdom; 3Applied Biosystems,
Darmstadt, Germany; kelly.warrington@eur.appliedbiosystems.com
Knockout mice
are important tools in
studying gene function and investigating genetic disorders as complete
loss of
gene function is established. A knockout mouse is generated by
replacing both
alleles of a target gene within an embryonic stem cell. This is done
using a
gene trap vector to generate a chimera (F1), selective breeding results
in F2
progeny and subsequent knockout mice (F3). At each stage a combination
of
genotypes is possible. The European Conditional Mouse Mutagenesis
programme
(EUCOMM) and the Knockout Mouse Project (KOMP), are utilising this
approach to
knockout ~ 20,000 genes, to provide a public resource of thousands of
knockout
mice by 2011. A major
challenge when generating
knockout mice is genotyping the F1-F3 progeny. The current method of
screening
these mice involves sequencing and long range PCR. The former technique
is time
consuming and laborious where as the latter technique is unable to
accurately
distinguish between the different genotypes. We have developed a rapid
and
accurate high throughput quantitative real time PCR method to determine
accurately genotype transgenic mice and their progeny. Furthermore,
this high
throughput approach can also be used to study human copy number
variation.
Genetic variation and in the human genome can cause susceptibility or
resistance to disease. Human copy number variation has been associated
with a
variety of diseases such as cancer, HIV infection, inflammatory
autoimmune
disorders, autism and schizophrenia. P007 TaqMan®
OpenArrayTM: A Breakthrough System For Nano-Well High Throughput
Genotyping 1Applied
Biosystems, part of Life Technologies, Molecular
research and analysis in
the field of plant and animal genomics is rapidly developing and
expanding into
new application areas, requiring the development and introduction of
new
technologies, assays and tools to support his research. Within this
broad field
of research, marker-assisted selection, QTL mapping and backcross
analysis are
widely used techniques to identify genes, loci and polymorphisms
encoded into
the genomes of all living organisms. Here we present the TaqMan®
OpenArrayTM
system, a breakthrough technology uniquely positioned for these kinds
of
applications. By combining gold standard TaqMan® SNP Genotyping
assays with the
efficient nano-reaction OpenArray™ platform, researchers are able to
genotype
large numbers of samples over any customizable set of markers with a
single
platform instrument. The TaqMan® OpenArray™ system utilizes 3072
nano-well
plates, innovative fluidic properties and a one day protocol to
generate high
quality, robust genotype profiles. This platform is especially enabling
for
researchers who have continuous streams of incoming samples that
require a
quick and cost-efficient workflow. To demonstrate the capabilities of
this
platform, we have genotyped several hundred single nucleotide
polymorphisms
across a panel of genomic DNA samples and report the performance in
terms of
assay pass rate, sample call rate and concordance to conventional
TaqMan® SNP
Genotyping. In addition, innovative solutions for research projects
with sample
quantity limitations have recently been developed and will be presented
here
for review. In summary, we will discuss how this system differentiates
itself
from the current genotyping technologies. P008 QuantPrime
- a flexible tool for reliable high-throughput primer design for
quantitative
PCR 1Potsdam
University, Germany; 2Max-Planck Institute for Molecular
Plant
Physiology, Potsdam, Germany; 3University of Silesia,
Katowice,
Poland; samuel.arvidsson@uni-potsdam.de
Medium- to
large-scale expression
profiling using quantitative polymerase chain reaction (qPCR) assays
are
becoming increasingly important in genomics research. A major
bottleneck in
experiment preparation is the design of specific primer pairs, where
researchers have to make several informed choices, often outside their
area of
expertise. Using currently available primer design tools, several
interactive
decisions have to be made, resulting in lengthy design processes with
varying
qualities of the assays. Here we present QuantPrime, an intuitive and
user-friendly, fully automated tool for primer pair design in small- to
large-scale qPCR analyses. QuantPrime can be used online through the
internet
(http://www.quantprime.de/) or on a local computer after download; it
offers
design and specificity checking with highly customizable parameters and
is
ready to use with many publicly available transcriptomes of important
higher
eukaryotic model organisms and plant crops (currently 295 species in
total),
while benefiting from exon-intron border and alternative splice variant
information in available genome annotations. Experimental results with
the
model plant Arabidopsis thaliana, the crop Hordeum vulgare and the
model green
alga Chlamydomonas reinhardtii show success rates of designed primer
pairs
exceeding 96 %. QuantPrime constitutes a flexible, fully automated web
application for reliable primer design for use in larger qPCR
experiments, as
proven by experimental data. The flexible framework is also open for
simple use
in other quantification applications, such as hydrolyzation probe
design for
qPCR and oligonucleotide probe design for quantitative in situ
hybridization.
Future suggestions made by users can be easily implemented, thus
allowing
QuantPrime to be developed into a broad-range platform for the design
of expression
assays. P009 Evaluation
of Digital PCR for Absolute Quantification LGC, Digital PCR
involves diluting and
partitioning a sample between many hundreds or thousands of individual
PCR
reactions such that a single molecule or less on average is present in
each
reaction. Determination of the number of positive amplifications is
indicative
of the number of targets present in the sample. As such, digital PCR
does not
suffer from inaccuracies that often arise from standard real-time PCR
approaches at trace levels using calibration curves, for example
through
extrapolation, and it affords the potential for absolute
quantification. Recent
advances in microfluidics have facilitated the development of digital
PCR by
combining microfluidics with single molecule, nanolitre volume PCR to
increase
levels of replication, throughput and cost efficiency. In this study
we have evaluated the
performance of digital PCR for absolute quantification using the
Fluidigm
BioMark Integrated Microfluidics System as compared to standard
real-time PCR
using the ABI 7900HT system. The performance of the systems was
evaluated using
a DNA standard of known molecular weight. The ability of the two
platforms to
discriminate changes in copy number was assessed using different ratios
of RNA
standards in a background of human total RNA. Sensitivity was
investigated
using different starting amounts of the RNA standards. The results
of this study are
presented here and demonstrate the potential of digital PCR approaches
for
absolute quantification. They also highlight some of the areas of
uncertainty
and variability that are inherent with this approach. Acknowledgements: This work was
supported by a grant
from the Department of Innovation, Universities and Skills (U.K.) under
the
National Measurement System Chemical and Biological Metrology Programme. P010 PERFORMANCE
OF THE THERASCREEN K-RAS MUTATION ASSAY ON THE LIGHTCYCLER480®Type
II
INSTRUMENT IN A CLINICAL ROUTINE SETTING Objectives : Recent
approval of Vectibix®
(Panitumumab) fully humanized monoclonal antibody therapy for advanced
colon
cancer created an urgent need in hospital laboratories to screen K-Ras
oncogene
mutations. We assessed the LightCycler® 480 instrument together
with the
Therascreen K-Ras mutation-specific scorpion primer PCR assay for its
practicality, easiness of use and performance in a medium sized routine
hospital laboratory setting. Methods : 40 genomic
DNA samples were
extracted from three 10 micrometer thick paraffin sections of
colorectal tumor
tissue using a QIAamp®DNA Mini Kit (QIAGEN). Prior proteinase K
digestion was
performed according the company instructions (DxS document GEN/023/2).
Each
section was pre-selected by a pathologist and checked for > 70%
tumor tissue
content. Final dissolved DNA content was measured with a Quant-IT dsDNA
BR
Assay Kit on a Qubit fluorometer (Invitrogen) or a Biophotometer
(Eppendorf). 20
ng purified DNA were used for the K-Ras Strip Assay™ (ViennaLab). 20 ng
purified DNA samples were used for the Therascreen K-Ras assay (DxS).
The
necessary assay adjustments for the LightCycler480®Type II
instrument are
described in the technical notes (DxS codes KR-03/04) and in the colour
compensation reagents instructions for use. Crossing points (CPs) were
determined with the second maximum derivative function of the absolute
quantification module (LightCycler® 480 software 1.5). Results : We
characterized PCR performance
parameters of the internal K-Ras exon 4 amplification control reagent
and two
external control genes (beta-actin and 18SRNA) on the
LightCycler480® system.
PCR efficiences on the formaldehyde-fixed and paraffin-extracted
genomic DNAs
(n=10) were high: beta-actin (1.93), 18SRNA (2.07), K-Ras (1.95).
Intra-assay
variabilities were small: beta-actin (CP: 31.73±0.81; CV=
<1.50), 18SRNA
(CP: 19.75±0.34; CV= < 0.31), K-Ras (CP: 28.47±0.52;
CV= <0.5). The
inter-assay variability (CV% of three assays performed within a month)
of the
mutation specific standard mixtures CPs within a Therascreen kit lot
(version
2) was as follows: Gly12Ala: 0.52, Gly12Asp: 0.92, Gly12Arg: 1.35,
Gly12Cys:
1.38, Gly12Ser: 2.2, Gly12Val: 0.24, Gly13Asp: 1.78. The results of the
DxS
real time PCR assay corresponded 100% with externally tested samples
(n= 20)
and with the K-Ras Strip Assay™ (n= 10). In two samples a double
mutation
(12Val/12Asp) or multiple mutations (12Ala, 12Cys, 12Ser, 13Asp) were
determined. Conclusions : The
Therascreen K-RAS assay
performed on the LightCycler480® instrument is a reliable technique
for
accurate mutation detection on paraffin extracted genomic tumor DNA.
Its major
advantages when performed within a clinical setting are the work time
savings
(no need for post-PCR processing) and a minimized risk for a laboratory
PCR
product contamination due to foil-sealed PCR plates. P011 Analysis
of gene expression profile by cDNA microarray and qPCR in fibroblast
cultures
of ALMS patients. 1Endocrine-Metabolic
Laboratory, Internal Medicine 3, Department of Medical and Surgical
Sciences,
University of Padua, via Ospedale 105, 35128 Padua, Italy; 2The
Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609, USA.; elisabetta.zulato@unipd.it
Introduction: Alström
Syndrome (ALMS) is a rare, autosomal monogenetic
disease, caused by mutation in ALMS1 (Chr 2p13), a gene
ubiquitously
expressed with unknown function. ALMS shows impairments at multiple
organ
systems, resulting in blindness, hearing impairment, childhood-onset
obesity,
hyperinsulinemia, insulin resistance and type 2 diabetes. A very common
feature
of ALMS patients is an extensive fibrosis evident at multiple
anatomical site:
kidney, heart, lung, liver, pancreas, bladder, ovary and testis. This
fibrotic
substitution can cause specific and severe organ failure bringing often
to an
early death. Aim: we
established fibroblast primary cultures from dermal biopsy of 4 ALMS
patients
and 3 control subjects. The cells were employed to analyze the
modulation in
gene expression profile related to the presence of mutated ALMS1 transcripts. Research design and methods: we
investigated in primary fibroblast cultures the gene expression profile
by cDNA
microarray analysis. RNA deriving from 4 ALMS patients was
co-hybridized with
the RNA pool obtained from 3 healthy controls, using an oligo-spotted
microarray platform. We quantified the collagen-specific genes
expression by
qPCR . Results and conclusions: From about
21500 genes represented, 188 resulted as up-regulated whereas 372 were
identified as down-regulated in ALMS patients. Gene function was
evaluated and
modulated transcripts were clustered in main categories such as
“extracellular
matrix component”, “cell cycle”, and “apoptosis”. Data analysis showed
an up
regulation of numerous collagen transcripts. The real-time PCR
quantification
confirmed ALMS fibroblasts express higher level of mRNAs coding for
different
type of collagens (in particular COL8A1 and COL15A1), suggesting an
involvement
of ALMS1 in the multi-organ fibrosis. P012 A
novel digital technology for non-enzymatic direct multiplexed
measurement of
gene expression 1NanoString
Technologies Inc., 201 Elliott Ave West, Suite 300, Seattle, WA 98119; 2The
Department of Microbiology, Box 358070, University of Washington,
Seattle WA
98195; 3Current address, Department of Bioengineering, Box
355061,
University of Washington, Seattle WA 98195; 4Division of
Biology
156-29, California Institute of Technology, Pasadena CA 91125; 5The
Institute of Systems Biology, 1441 N. 34th St., Seattle WA 98103; ramin@novoptim.com We describe a
novel technology, the
nCounter system, for highly multiplexed analysis of gene expression
levels.
Then nCounter system detects individual mRNA molecules using an
assigned code
sequence of fluorescent molecules, and counts the number of times that
code
appears in a sample. No enzymes are used in our system; rather, the
collection
of probes is hybridized in solution to RNA in a sample. Experiments
performed
in a single multiplex analysis of 550 human genes revealed a
correlation
coefficient of 0.999 between replicate measurements, a detection limit
between
0.1fM (0.2 copies/cell) and 0.5fM (1 copy/cell), and a linear 500-fold
dynamic
range. The nCounter system can detect a 1.5-fold increase or decrease
in
expression across a broad range of expression, and as little as 20%
changes in
expression for genes present between 1fM and 10fM. We demonstrate a
good
correlation between nCounter system and Affymetrix GeneChip technology,
and
better correlation with TaqMan, for –fold change measurements using two
different experimental paradigms. Furthermore, a comparison of
transcript
levels measured by the nCounter system with SYBR green RT-PCR
demonstrated a
high correlation in the gene expression pattern at all transcript
levels. We
show that a whole cell lysate can be used as starting material with
equivalent
results to purified total RNA. Finally, we show that RNA extracted from
formalin-fixed paraffin embedded (FFPE) tissues can be used in the
nCounter
system to analyze expression levels in archived samples. Our unique
direct
detection and digital quantification approach results in unprecedented
sensitivity,
precision and reproducibility in gene expression analyses. Materials
&
Methods - nCounter hybridization reactions were performed in triplicate
with
total RNA samples isolated from mock and polio virus infected human
A549 cells.
nCounter reactions were set up as follows: 100ng of total RNA Reporter
and
capture probes for 509 human mRNAs and controls made to non-human
sequences (6
positive, 2 negative) DNA control targets spiked in at 0.1, 0.5, 1, 5,
10 and
50 fM Hybridizations were carried out for 20h at 65°C. Excess
reporters were
then removed by using magnetic bead based purification. The same
samples and
amount of RNA were also analyzed with Affymetrix® U133Plus2 arrays,
using the
two-cycle amplification/labeling protocol recommended by the
manufacturer. We
selected a subset of 14 genes in which the measured log2 fold-change
was
significant in one platform but not the other for further analysis by
TaqMan
Real-Time PCR. In a second experiment, nCounter hybridization reactions
were
performed in triplicate as described above with total RNA samples
isolated from
sea urchin embryos collected at seven different development time
points. A set
of 21 genes were selected for comparison with existing SYBR Green
Real-Time PCR
data generated in the Davidson Lab. P013 DNA
amplification in flow-through microreactor 1Institute
of Photonic Technology, The work
presents a microfluidic
chip system with optimized thermal and fluidic characteristics for
flow-through
polymerase chain reaction. The designed microreactor comprises a
heating plate
consisting of 5 temperatures zones and a fluidic chip with meandering
microchannels. The optimized thermal profile allows the implementation
of one
PCR cycle in a half channel loop. With this condition the microreactor
possesses a 40 cycles flow-through thermocycler on the footprint of a
microscope slide. In addition the microfluidic chip system was designed
to
operate at segmented-flow conditions for high-throughput analysis of
PCR
samples in a small volume of 10 – 100 nl. Each droplet of PCR solution
in a
flow of mineral oil may contain a single sample that is independently
processed
while transported through the microchannel. The surface of the
microchannels
was chemically modified to assure stable fluidic conditions. The PCR
conditions
can be adapted to extensive applications by variation of the flow-rate
and the
geometry of the temperature zones. The system is designed for an
application in
point of care tests and as a part of a system for the control of a
fermentation
process. Due to the sensitivity of the PCR process to contaminations
the use of
disposable microchannel devices is commonly preferred. Disposable chip
devices
made from polycarbonate were tested within the flow-through
microreactor for
the detection of the tumor suppressor gene p53 and compared to the
functionality of all-glass chip devices. Although product yield and
selectivity
of the PCR process do not depend on material of the microchannel
devices, a
well defined and reliable segmented flow could only be realized in the
all-glass microchannel device. The MINAMED-project is supported by
grants of
the BMBF (16SV3529). P014 PCR
and microarray chip technologies for Phytophthora diagnosis 1Institute
of Photonic Technology, The
polymerase chain reaction (PCR)
has been established as a standard method in molecular biological
analysis for
amplification of small amounts of nucleic acids. Due to the
amplification of
the target DNA their detection becomes easier or even only possible.
Therefore,
electrophoresis, real-time PCR or hybridization based assays using DNA
microarray technology are applied. Recently, we
developed a stationary
PCR chip device for fast DNA amplification using minimal volumes of
reaction
mixture with low power consumption. For example the amplification of a
131 bp
fragment can be performed within 23 min including 45 PCR cycles.
Heating and
cooling ratios of up to 15 K/s are realized and the power demand
amounts to
approximately 3 watts. The rapid operating speed results from low
volumes of
reaction mixture (down to 0,5 µl) that are applied on the chip
surface where
thin film platinum layers are located that act as heating structures
and
temperature sensors. Special disposable glass slides are utilized as
sample
carriers to realize single use only in order to avoid cross
contamination. A
miniaturized device including the stationary PCR chip and all elements
for
control is already developed. This portable device includes a PDA
(personal
digital assistant) for parameter input and control as well as
rechargeable
batteries that provide sufficient power for one day of operation. A
fibre-optics based extension is available for real-time monitoring of
the
amplification process that enables quantization of the target DNA.
Thereby time
consuming DNA analysis following the amplification steps are
eliminated. For
this purpose different fluorescence based detection methods with SYBR
Green as
intercalator or a specific TaqMan probe were applied. Using the
stationary PCR
chip a total amount of only 2 DNA-molecules/µl could be
reproducibly detected. Current
and future works are focused on integration of the PCR process and
DNA-chip-technology for readout of information using a single device.
Caused by
the disadvantages of fluorescence readout such as complicate detection
equipment that is also bulky and expensive we use a reliable electrical
detection system based on enzymatically catalysed, reductive silver
enhancement
that bridges microelectrodes. For this purpose a DNA chip with
integrated
electrode gaps was developed. Our aim is the development of an
analytical
system for point-of-care diagnosis of the phytopathogen Phytophthora
that
causes decay of several plants such as soya, oak, potato or tomato. This work is
supported by grants of
the Federal Institute of agriculture and food (support code:
28-1-42.027-06). qPCR NOS Session
Oligo
Design Across the Mouse Genome Biosearch
Technologies, Inc., Fluorescence-quenched
probes are
routinely used to gauge gene copy number. We describe a bioinformatic
engine
for the design of such oligos, and used to generate five thousand
TaqMan assays
for the NIH Knockout Mouse Project (KOMP). Here, we demonstrate the
performance
of a subset when amplified upon wild-type mouse gDNA. Analysis of this
data-set
uncovers important trends in amplification performance and emphasizes
the need
to screen assay specificity using both bioinformatic and empirical
approaches.
Redundancy and accessibility are considerations that become pronounced
in
large-volume sequence design. Based on this experience as well as user
feedback, new software functionality is introduced to improve upon
these
qualities. P016 NIFES, Background:
Extensive sequencing
efforts have been taking place for the Atlantic cod (Gadus morhua) in
recent
years, the number of ESTs in the Genbank has reached more than 140.000.
Despite
its importance in Results: The
stability of 10 potential
reference genes was examined in six tissues of Atlantic cod obtained
from four
populations, to determine the most suitable genes to be used in qRT-PCR
analyses. Relative transcription levels of genes encoding β-actin
(ACTB),
elongation factor 1A (EF1A), actin-related protein-2 (ARP-2),
glyceraldehyde-3P-dehydrogenase (GAPDH), ubiquitin (Ubi), acidic
ribosomal
protein (ARP), ribosomal protein S9 (S9), ribosomal protein L4 (RPL4),
RPL22
and RPL37 were quantified in gills, brain, liver, head kidney, muscle
and
middle intestine in six juvenile fish from three wild populations and
from
farmed Atlantic cod. Reference gene stability was investigated using
the geNorm
and NormFinder tools. Based on calculations performed with the geNorm,
which
determines the most stable genes from a set of tested genes in a given
cDNA
sample, ARP, Ubi, S9 and RPL37 were among the most stable genes in all
tissues.
When the same calculations were done with NormFinder, the same genes
plus RPL4
and EF1A were ranked as the preferable genes. Conclusions:
Overall, this work
suggests that the Ubi and ARP can be useful as reference genes in
qRT-PCR
examination of gene expression studying wild populations of Atlantic
cod. P017 Validation
of housekeeping genes for gene expression studies in human ejaculate Department of
Urology and Pediatric
Urology, Justus Liebig University Giessen, Germany; marcia.c.oliveira-cavalcanti@vetmed.uni-giessen.de
BACKGROUND:
Beta-actin,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), heat shock protein
(HSP) and
ATP-synthase subunit 5B (ATP5b), with distinct functional
characteristics and
expression patterns were analyzed in use as references for gene
expression
profiling using quantitative Real-Time PCR (qRT-PCR) in human
ejaculate.
OBJECTIVES: To determine the expression stability of 4 commonly used
reference
genes in the ejaculate of fertile and infertile patients. METHODOLOGY:
Semen
was evaluated using standard World Health Organization (WHO)
procedures. Each
assay included a standard curve (exponent base 10) of four serial
dilution
points of cDNA (raging from 5 ng to 0.005 ng). All samples were run in
duplicate and the mean value of each duplicate was used for all further
calculations. The stability of selected reference genes was analyzed
using the
GeNorm software. RESULTS: Calibration curves were generated using
relative
concentrations vs. threshold cycles (Ct). The RSq value (R2, linear
correlation
coefficient), an indicator of fit for the standard curve plotted to the
standard data points of all genes ranged from 0.982 to 1.000. Based on
the slopes
of the standard curves, the mean of the amplification efficiencies were
above
97%. The genes showed different expression between fertile and
infertile
patients. The Ct deviation averages were GAPDH (2.17), beta-actin
(2.32), HSP
(1.053), and ATP5b (3.34). GeNorm identified GAPDH and beta-actin as
the most
stable pairwise variation of reference genes for the infertile
ejaculates (M
value for combination of best two genes were 0.148). CONCLUSION: In
infertile
patient samples, a combination of GAPDH and beta-actin yields stable
reference
gene expression levels, whereas the use of HSP or ATP5b is not suitable
for
normalization of qRT-PCR results in this sample type. Our results
provide
information an appropriate reference gene for the normalization of
qRT-PCR data
in the ejaculate necessary for future gene expression studies. P018 Importance
of RNA integrity assessment in a qRT-PCR workflow Agilent
Technologies, Real-time
quantitative PCR (QPCR) is
a highly sensitive method to assess gene expression changes in
biological
systems. As for all experimental designs, high quality starting
material is
essential for the success of the experiment. There are various
mechanisms by
which RNA can be degraded either at the 5’ or 3’ end. Not knowing the
extent of
possible degradation can lead to false negative results or
misinterpretation of
data if the amplicon falls into a degraded region. Therefore, the
degradation
level of RNA samples is an important parameter to monitor when
designing
primers and probes for QPCR. Here, it's shown how on-chip
electrophoresis
combined with a special RNA Integrity Number (RIN) algorithm can be
used to
assess the level of degradation of the RNA starting material. The
results of
the experiments indicate that the amount and directionality of
degradation are
highly gene-dependent and that the most pronounced effects appears
below a RIN
of 4.6. P019 1Synlab
Medical Care Service, Germany; 2Institute of Human
Genetics,
University of Regensburg, Germany; 3Applied Biosystems,
part of Life
Technologies, Foster City, USA; 4Applied Biosystems, part
of Life
Technologies, Germany; Astrid.Ferlinz@eur.appliedbiosystems.com
Cellular
lipidomics is defined as
the analysis of metabolism, transport, and localization of lipids
species
within cells. The quantitation of different lipid species from various
biochemical pathways and biochemical analysis of lipid metabolism
enzymes is an
integral part of this concept. Recent progress in the field of
transcriptomics,
mainly the cost reduction of DNA-microarrays and the development of
high-throughput real-time reverse-transcription (RT)-PCR systems have
also
enabled researchers to perform a comprehensive transcriptomic analysis
of all
lipid-related genes. Here we
describe the quantitative
analysis of 41 selected lipid-related transcripts using a novel
“Lipidomic”
TaqMan Array. The TaqMan Array is based on an Applied Biosystems 7900HT
microfluidic card. This method allows simultaneous analysis of 41
lipid-related
genes and 7 controls in 2 replicates of 4 different samples per run. In
addition, we analyzed the identical “Lipidomic” gene set and identical
RNA
samples on the recently launched “TaqMan Express Plates” – customizable
96-well
plates with pre-spotted TaqMan Gene Expression Assays. Our special
interest was to study
the expression of “lipidomic” genes in macrophages and microglia under
conditions mimicking sterol loading and pro-inflammatory activation. The TaqMan
Array results show that
(i) stimulation with the liver-X-receptor (LXR) and retinoid-X-receptor
(RXR)
ligands T0901317 and 9-cis retinoic acid (RA) induces several genes of
lipid
metabolism, (ii) lipopolysaccharide (LPS) and interferon-g (Ifn-g)
strongly
repress lipid-related genes, and (iii) co-incubation with
docosahexaenoic acid
(DHA) dampens the repressing effect of LPS. Our results were confirmed
by the
data obtained with the TaqMan Express Plates. The method
described here can be
used to rapidly and accurate quantify transcriptionally dynamic “lipid”
genes
in any cell type. The “lipidomic” TaqMan Assay Set may be applied to
study
lipid disorders or to quantify the transcriptional effects of
pharmacological
treatments on lipid-related genes. P020 1Bio-Rad
Laboratories, Inc., 6000 James Watson Drive, Hercules, CA 94547; 2Bio-Rad
BioRecherche, 3 Blvd R. Poincare, 92430 Marnes la Coquette, France; arnaud.remy@bio-rad.com RNA quality
plays a major role in
the generation of accurate quantitative results from gene expression
analysis
experiments. cDNA made from RNA that has been degraded will not become
amplified to the same degree as cDNA made from intact, undegraded RNA.
This can
lead to erroneous conclusions regarding levels of gene expression when
comparing samples that are degraded to different extents. To examine
the
effects of RNA degradation on quantitation of specific gene
transcripts, qPCR
was performed on equivalent amounts of RNA that had been degraded to
various
extents. The detection of amplified product was seen at successively
later
cycles as the RNA was degraded over time. The CT values of the qPCR
reactions
from five gene transcripts (18S rRNA, β-actin, β-tubulin, HPRT and
GADPH)
showed different degradation rates. Comparing qPCR results derived from
RNA in
different states of degradation will generate very different
quantitative
conclusions. This can be as great as 1000 fold, with samples subjected
to 7 hr
of heat degradation. The Experion automated electrophoresis system
(Bio-Rad
Laboratories, Inc.) provides an effective method for determining both
the
quality and quantity of RNA in gene expression analysis experiments
using as
little as 200 pg of total RNA - several thousand times less material
than that
required for gel electrophoresis. The calculation of the RQI uses an
algorithm
that compares three regions of an electrophoretic profile, with
differential
weighting, to a series of degradation RNA standards scale from 10
(intact) to 1
(fully degraded). The very simple concept behind the RQI gives results
that are
comparable to the RIN. The RQI is accurately calculated over a wide
range of
RNA concentrations (200 pg to 500 ng), is very reproducible (%CV
<3), and is
applicable to a wide range of mammalian tissues. The RQI number by
itself is
not sufficient to decide if a sample can be safely used for downstream
application. The user must determine empirically what threshold their
specific
samples require. An upfront validation process is required to correlate
RQI
values and successful or failed downstream application, using a set of
artificially degraded samples. This process will allow defining
threshold RQI
values. The Experion software groups the samples into three colour
codes, which
appear in a summary screen for a quick visualisation. Once the
threshold values
have been determined, it is possible to customize the range for each
colour
group. We show that quantitation of RNA using qPCR correlates well with
RQI
measurements. By providing an RQI score and electropherogram, the
Experion
automated electrophoresis system allows even the most inexperienced
user to
quickly and effectively quantitate the level of degradation of an RNA
sample in
a systematic manner in order to reliably detect differences in gene
expression
using RT qPCR experiments helping ensure reliability in results. P021 Application
to RAM Amplification of Real-time Analysis Methodologies Developed for
PCR The use of
model-based amplification
kinetics parameter estimates may improve the accuracy and productivity
of
real-time amplification by extracting more information from each
experiment.
Algorithms implemented for PCR analysis can be usefully applied to
2-primer
ramified single-stranded circle amplification (RAM.) Although RAM
and PCR kinetics are
sufficiently similar to be analyzed with implementations of the same
algorithms
there are essential differences between the two technologies. One
significant
difference for kinetic analysis is that the isothermal RAM reaction can
be
sampled at a higher frequency than the PCR, as PCR is limited to one
data point
per cycle (a cycle can be sampled multiple times but those samples aim
toward a
single point-estimate.) By contrast, RAM kinetic data can be collected
continuously (limited only by instrumentation.) Greater sampling
density allows
more precise identification of kinetic phase transition (e.g. baseline
to
exponential phase, exponential to linear phase.) Here the
application of kinetic
parameter identification to RAM amplification is shown, and compared to
analogous PCR analysis. While fitting parameterized models to RAM
kinetics is
done as for PCR, the interpretation of a RAM amplification fitted model
is
analogous but distinct from the interpretation of a PCR model. For
example, PCR
efficiency (signal increase per cycle) doubles at its theoretical
maximum; in
RAM the analogous interpretation is signal increase per time unit, and
measures
the rate of the reaction. RAM
amplification, like the PCR, can
be used in high-throughput diagnostic assays. It is hoped that a more
quantitative understanding of the RAM reaction will encourage broader
application of the technology. P022 1Laboratory
of Nutritional Genomics, Institut Polytechnique LaSalle Beauvais,
France; 2Animal
Science Department, Faculty of Agricultural and Food Sciences, American
University of Beirut, Lebanon; afif.abdelnour@lasalle-beauvais.fr
We have
developed and validated an
alternative method of the absolute quantitative real-time PCR based on
the use
of plasmid. Our method uses a Bacterial Artificial Chromosome vector
pBeloBAC11. In contrast of plasmid, pBeloBAC11 is present in a single
copy
number in the bacterium E. coli EC100. Taking benefit of that we
constructed a
reliable standards curve based on initial input amount of BAC vector
harboring
a single copy of the human Reduced Folate Carrier transcript (hrfc) and
the
Folate Binding Protein transcript (fbp). Standard curves for each assay
were
highly reproducible with no significant difference in slopes between
three
different runs of the three different assays. The dynamic ranges were
wide,
ranging from 1x102 to 1x107 copies. The linearity R2 coefficient of Ct
was 0.99
for the recombinant BAC. Q-PCR efficiencies were 0.991 (CV=0.09%) and
0.992
(CV=0.06%) for hrfc and fbp, respectively. The method has been applied
for
simultaneous quantification of the hrfc and fbp transcripts in tumor
tissues
and in their matched adjacent normal tissues. The method is sensitive
and
produces quantitative data with a good efficiency. It may be used
routinely for
measuring multiple gene expression in diseases evolution. P023 Removal
of contaminating genomic DNA in QRT-PCR using a shrimp nuclease. Thermo Fisher
Scientific, ABgene
House, DNA
contamination can often occur in
quantitative reverse transcription – polymerase chain reactions
(QRT-PCR), and
should be removed in order to avoid false positive results. DNase I is
commonly
used for removing DNA contamination, but this has a relatively long and
harsh
protocol which introduces an extra step between the isolation of RNA
and the
QRT-PCR reaction itself, as well as increasing the risk of RNA
degradation due
to the harsh inactivation conditions. A nuclease from the arctic shrimp
Pandalus
borealis , has properties that make it useful for the removal of
contaminating DNA. The nuclease activity of the enzyme is specific to
double
stranded DNA, which therefore allows the enzyme to be added directly
into the
reverse transcription step. Unlike DNase I, the shrimp nuclease is
easily
inactivated at high temperatures, such as those used for the RT
deactivation/hot start incubation step of a QRT-PCR. Here we show how
the
addition of shrimp nuclease during the reverse transcriptase step can
remove
contaminating DNA from the reaction. Human genomic DNA (100ng -10pg)
was
incubated with or without shrimp nuclease (10 – 0.1 units) before
amplification
of a 74bp fragment of the Apolipoprotein B gene was carried out. The
percentage
removal of genomic DNA was calculated by comparing the delta Ct values
between
reactions where the DNA was pre-incubated with shrimp nuclease to
reactions
incubated without shrimp nuclease (control). In order to determine
whether the
Ct shift was caused by inhibition of QPCR by shrimp nuclease, the same
units of
pre-inactivated shrimp nuclease was added to a separate reaction.
Whilst large
Ct shifts were observed at all DNA concentrations for 10-1 units of
shrimp
nuclease, there was also significant inhibition caused by the addition
of
inactivated enzyme at these concentrations. Therefore, not all of the
Ct shift
can be attributed to removal of contaminating DNA. However, at
concentrations
of between 0.8 – 0.4 units, there was no inhibition observed by
inactivated
shrimp nuclease and between 99.4% - 100% removal of the DNA was
observed
depending on the concentration. Therefore, at 0.8 – 0.4 units this
enzyme
effectively removes any contaminating DNA without adding an extra step
to the
QRT-PCR protocol - and completely eliminates the need for DNase I
treatment. In
so doing, it increases the accuracy and reproducibility of QRT-PCR
reactions,
especially when using crudely purified samples. P024 The
effect of amplicon characteristics on the success of fast QPCR. Thermo Fisher
Scientific, ABgene
House, Adapting QPCR
experiments to run
using fast cycling conditions is a simple method of increasing
experimental
throughput by reducing run duration times by up to 50%. QPCR results
and the
quality thereof can be greatly affected by the characteristics of the
product
being amplified. This is especially true when employing fast thermal
cycling
protocols, which have been shown to reduce the sensitivity and increase
variability of some assays. Speeding up thermal cycling protocols can
lead to
reaction failure if attempting to amplify a non-optimal assay or
difficult
target. Therefore we have investigated the effects of amplicon length,
GC
content and secondary structure, on the performance of fast QPCR
experiments
compared to when a standard thermal cycling protocol is used. A panel
of human
specific assays were employed in QPCR experiments using fast and
standard
cycling protocols. The assays were designed so that the resulting
amplicons had
a broad range of length, GC content and secondary structure (minimum ΔG
values
calculated at 60C). Experiments were carried out on the Roche
LightCycler 480
and assay sensitivity was assessed by comparing delta Cp values,
standard
deviations and differences in QPCR efficiency (calculated by the
standard curve
method). The results demonstrate that amplicons that are of excessive
length,
high in secondary structure or high in GC content can all be causes for
poor
fast QPCR results. Therefore, we recommend that assays be designed with
these
amplicon characteristics taken into account, in order to ensure
successful fast
QPCR experiments. P025 The
effect of white pigmentation in 96-well plates during QPCR. 1Thermo
Fisher Scientific, ABgene House, Blenheim Road, Epsom KT19 9AP. United
Kingdom; 2School of Life Sciences, Oxford Brookes
University,
Gypsy Lane,
Oxford OX3 0BP. United Kingdom; ian.kavanagh@thermofisher.com
QPCR
instruments are able to monitor
amplicon quantity in real-time during PCR reactions by detecting
fluorescence
signals and recording fluorescence data. The majority of fluorescence
is
reflected out of the PCR tube either by the polypropylene itself or by
the
walls of the thermal cycler block, when clear polypropylene is used. Aims: To investigate if white 96-well plates
allow better fluorescence detection of PCR products during QPCR assays
than
clear 96-well plates. Methods: QPCR experiments were performed by amplifying target
segments of
the genes sigB , dnaK , srfAA , and argB
from the
genome of Bacillus subtilis BBK006. SYBR Green I was used to
monitor
product accumulation via fluorescence. To examine the effect of
pigmentation on
CT, 96-well plates were custom-manufactured with elevated pigment,
reduced
pigment or differently-compounded white polypropylene. Results: In general
QPCR reactions on white
plates have lower CT values compared to QPCR reactions on clear plates.
Statistical analysis revealed there was no significant difference
between CT
values of sets dnaK and argB when using clear and
white plates.
However, analysis of melt curves suggested this was due to poor primer
hybridisation. Further analysis revealed that CT values of sets srfAA
and argB were significantly lower when using white
plates
compared to clear
plates. There was also a significant difference between melting curves
of low
template DNA reactions when using clear and white plates. Conclusions:
It was observed that signal noise was more prevalent in the melting
curves of
reactions using clear plates than white plates. There were no
significant
differences between CT values and amplification plots of low template
DNA
reactions when using the standard white plate compared to plates with
altered
pigmentation. This, coupled to examination of the plates by scanning
fluorimetry, suggests that the effect of the pigment is complex and not
necessarily linked to reflection of signal alone. P026 Alignment
of the heat shock protein gene (hsp) sequences and development of
multiplex PCR
method for the simultaneous detection of bovine mastitis pathogens
including
Staphylococcus aureus, and Streptococcus spp. Bovine
mastitis is a multifactorial
disease caused by many different bacteria species. Of these bacteria
species,
Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis,
and
Streptococcus bovis are the major pathogens. Since the heat shock
protein (hsp)
genes, especially hsp60, has been shown to have more discriminatory
power than
16S rRNA gene and inter-transcriptional spacer (ITS) region, in this
study, we
tried to develop a multiplex PCR method based on the hsp (heat shock
protein)
genes for the specific detection of S. aureus, S. agalactiae, S.
uberis, and S.
bovis. Molecular weight of the PCR products amplified were 406 bp,
350bp,
119bp, and 247bp, respectively, for S. aureus, S. agalactiae, S.
uberis, and S.
bovis. Using this multiplex PCR method, all the selected target strains
could
be specifically detected in food samples. As the multiplex PCR method
was used
for direct detection of mastitis pathogens in milk samples, the
detection limit
was N (N= 1–9) ×103 CFU/ml of milk samples. If a 10 h
pre-enrichment step was
performed, the detection limit was N×100 CFU/ml. Thus, the
multiplex PCR method
could be used for the specific and sensitive detection of these
pathogenic
bacteria in food and milk samples. P027 qPCR
as a method to estimate synchronization of immortal Hepa 1-6 cells –
problems
with inhibitors and low abundant genes The aim of
our experiment was to
synchronize the mouse immortal hepatoma cells Hepa 1-6 and to prove the
success
of synchronization by measuring the expression of representative genes
from
cholesterol synthesis and circadian regulation. These genes are known
to be
expressed in a circadian manner in the mouse liver. However, many of
them are
expressed at low levels in immortal cell lines which represents a
challenge for
their quantification. Hepa 1-6
cells were grown in 12-well
plates until confluency. Cells were treated with 10 uM forskolin,
washed twice
with ice cold PBS, lysed with TRI reagent (Invitogen) and stored at
-80ºC until
RNA isolation was performed according to TRI reagent protocol. Pelet
was washed
once with 75% etanol and resuspended in RNAse free water. Because of
low RNA
yield cDNA was synthesized starting from 200ng of total RNA which is a
5 times
lower concentration as usual. qPCR reaction efficiency was evaluated
with
serial dilutions of cDNA with mCyp51 (a gene from cholesterol
synthesis) and
18sRNA primers on Light Cycler 480 (Roche) using the cyber green
approach. The
results showed that several dilutions result in the same Cp value,
while at
some dilutions there was no amplification at all. After testing
different
serial dilutions the expected results were obtained only if cDNA was
diluted to
a specific (narrow range) concentration. To increase RNA amount samples
with
the same treatments were pooled, dried and precipitated with sodium
acetate.
The qPCR has been repeated with serial dilutions and the expected
amplification
pattern was observed (different Cp values according to dilutions). It was
concluded that the original
samples contained an unidentified inhibitor of either qPCR or cDNA
synthesis
that has been removed by drying RNAs followed by another precipitation.
After
solving this problem we measured the timely expression of Cyp51,
Dbp and Bmal1 in a time-series experiment spanning 48
hours. In
the first 8h
-12h forskolin provokes an immediate early response of the 3 measured
genes.
According to the expression of Dbp that is abundant also in
immortal
cells, we conclude that synchronization of Hepa 1-6 cells line has been
successful
while the results for Cyp51 and Bmal1 are not
conclusive.
Additional optimization procedures are required to enable quantitative
time-series measurements of low expressed genes in immortal cells. P028 Automated
extraction of viral RNA from blood samples for Bluetongue virus
diagnosis Veterinary
and Agrochemical Reseach
Center (VAR), Objectives -
Bluetongue (BT) is a
non-contagious, insect-borne disease caused by a virus from the genus
Orbivirus
within the family Reoviridae. The presence of viral RNA in clinical
samples is
monitored by silica-based nucleic acid purification followed by
real-time reverse
transcription PCR (RT-qPCR) analysis. This study describes a
comparative
validation of a manual and an automated silica-based procedure for the
extraction of nucleic acids from whole blood samples. Methodology
and results - First, the
linearity of the automated assay was assessed by testing a 10-fold
dilution
series of spiked blood with a viral load ranging from -0.32 to 5.7
log10 TCID50
ml-1. The linearity was analysed by linear regression and ranged from
-0.32 to
5.7 log10 TCID50 ml-1. The limit of detection (LOD) of the automated
assay was
determined by analysing a 2-fold dilution series of spiked blood
samples.
Probit analysis was used to calculate the input concentration with a
95%
probability of a positive RT-qPCR result which yielded an LOD of -1.32
log10
TCID50 ml-1. The intrarun and interrun variability of the extraction
protocol
is an important characteristic of the automated extraction protocol
which can
extract up to 184 blood samples in a single run. The intrarun and
interrun
variability were assessed by extracting a 1:2 dilution series of spiked
blood
with a viral load ranging from -1.15 to 2.46 log10 TCID50 ml-1. Each
dilution
was extracted automatically 5 times in each of 5 independent runs. The
coefficient of variation (%) was calculated for each dilution and
ranged from
1.26 to 7.26 with an increase in variation towards the LOD. A
comparative
analysis of both extraction protocols was made by analysing 50 field
samples
from the 2008 BTV epizootic that were extracted both manually and
automatically.
Statistical analysis using Pearson’s coefficient, Bland-Altman analysis
and
Passing-Bablok regression analysis, all indicated that the mean
difference
between both procedures is negligible (-0.37) and that most results
fell within
the 95% confidence interval of the mean (95% CI: 0.64 to -0.10). To
validate
the absence of cross-contamination, 48 strongly positive and 48
negative blood
samples were arranged in a checkerboard pattern. The 96 samples were
extracted
automatically and evaluated for cross-contamination by RT-qPCR
analysis. No
viral RNA could be detected in any of the 48 negative samples. Finally, the
throughput and
hands-on-time of the automated extraction protocol were estimated. The
automated purification of nucleic acids from 184 blood samples took 2
hours 30
minutes. Beside the important increase in capacity, the automated
protocol
requires much less hands-on-time resulting in a more efficient flow
from sample
to RNA and from RNA to qRT-PCR. Conclusion -
In conclusion, our
findings clearly demonstrate that both extraction protocols give
essentially
the same results and that both protocols can therefore be used
interchangeably. P029 Validating
internal controls for Cucurbita pepo real-time PCR studies 1IFAPA
Centro “Alameda del Obispo”, Área de Mejora y
Biotecnología. Avda. Menéndez
Pidal s/n 14080 Córdoba, Spain.; 2IFAPA Centro “La
Mojonera”, Área
de Mejora y Biotecnología. Autovía del
Mediterráneo sal. 420. E-04745, La
Mojonera, Almería, Spain.; clarai.gonzalez@juntadeandalucia.es
Members of
Cucurbitaceae family
(pumpkin, squash, cucumber and watermelon) make a significant
contribution to
our intake of vitamins and minerals. Among them, squash ( Cucurbita
pepo )
is an economically important species because of its nutritional
quality,
relative low price and year-round supply. Several studies have been
developed
in squash in order to study both quality and stress response aspects.
Understanding patterns of expressed genes during squash development may
provide
insight into complex regulatory networks and could contribute to the
breeding
process of the species. Real-time PCR
is the most used
method to quantify biological changes in mRNA levels. In order to
control
several variables in real-time PCR experiments, proper reference genes
are
commonly used (Vandesompele et al. , 2002). A need for
reference genes
in C. pepo has emerged and the studies in this area have not
yet been
evaluated. For this reason, we have carried out an extensive evaluation
using
the BestKeeper program (Pfaffl et al. , 2004) with the aim of
studying
the transcripts stability of eight commonly used housekeeping genes in
thirteen C. pepo samples. This work
presents the details and
findings from our validation of 18S, elongation factor, actin, tubulin,
ubiquitin, protein phosphatase 2A, helicase and
glyceraldehyse-3-phosphate
dehydrogenase genes in different C. pepo tissues and time
points (root,
leaf, shoot, flower and fruit) as well as under salinity, cold and
drought
stresses. The normalization strategy presented here is a prerequisite
to
accurate real-time PCR expression profiling that opens up the
possibility of
developing reliable experiments in squash. References Pfaffl MW,
Tichopad A, Prgomet C,
Neuvians TP. 2004. Determination of stable housekeeping genes,
differentially
regulated target genes and sample integrity: BestKeeper – Excel-based
tool
using pair-wise correlations. Biotechnology Letters 26: 509–515. Vandesompele
J, De Preter K, Pattyn
F, Poppe B, Van Roy N, De Paepe A, Speleman F. 2002. Accurate
normalization of
real-time quantitative RT-PCR data by geometric averaging of multiple
internal
control genes. Genome Biology 3, 7. P030 Indentification
of stable reference genes for spatial and temporal gene expression
studies in
human brain. Identification
of stable reference
genes for spatial and temporal gene expression studies in human brain. Runa M.
Grimholt, Marianne Kringen,
Reidun Øvstebø, Jens Petter Berg & Armin P. Piehler R&D-Group,
Department of
Clinical Chemistry, Background: Quantitative
real-time RT-PCR
(qRT-PCR) is a powerful tool and widely used for gene expression
studies.
Internal reference genes, so-called housekeeping genes, are mainly
employed to
normalize mRNA levels between samples. However, several studies have
shown that
expression of traditional housekeeping genes may vary substantially
under
certain conditions. Therefore, validation of stable reference gene
expression
is mandatory in mRNA quantitation studies using qRT-PCR. In our study,
we
sought to identify stably expressed reference genes in human brain
tissue from
several brain regions and different developmental stages. Methods -
Traditional housekeeping
genes and genes recently proposed to be stably expressed under certain
conditions were chosen from the literature. Expression of altogether
twelve
candidate reference genes were assessed in tissues from 35 different
adult and
7 fetal brain regions. To calculate the most stably expressed genes and
the
recommended number of reference genes required for qRT-PCR
normalization, data
were analyzed using the software package geNorm. Results - In
the group comprising
adult brain tissues from 35 different regions, the genes encoding PGK1
(phosphoglycerate kinase 1), TBP (TATA box binding protein) and SDHA
(succinate
dehydrogenase complex, subunit A) showed least variation in mRNA
levels. In 7
fetal tissues from different brain regions, the genes SDHA, ACTB
(actin, beta)
and CTBP1 (C-terminal binding protein 1) were the most stably expressed
genes.
The genes encoding PGK1, SDHA and CTBP1 showed highest stability across
the
different developmental stages. Interestingly, b-2-microglobulin (B2M)
and
ubiquitin C (UBC), two genes frequently used to normalize qRT-PCR data,
exhibited the highest variation of all tested candidate reference genes
in all
three brain tissue groups. Conclusion -
Employment of several
housekeeping genes is needed for data normalization in qRT-PCR studies
on
different human brain regions and developmental stages. When studying
spatial
gene expression in human brain, the housekeeping genes PGK1, TBP and
SDHA are
recommended for normalization of expression levels in adult brain
tissues, and
the genes SDHA, ACTB and CTBP1 for fetal tissues, respectively. For
temporal
gene expression studies, the combination of PGK1, SDHA, CTBP1 and
GOLGA1 (golgi
autoantigen, golgin subfamily a, 1) seems to be suitable for qRT-PCR
normalization. P031 Selection
of suitable reference genes for qRT-PCR for the study of circadian gene
expression in mouse liver. 1Center
for Functional Genomics and Bio-Chips, Faculty of Selection of
appropriate reference
genes is an important step in qRT-PCR that ensures accurate analysis
and
normalization of data generated. Circadian gene expression studies
carried out
by qRT-PCR have often used normalization based on one or two reference
genes.
18sRNA and ß-actin are considered as housekeeping genes, though
their
expression has been shown to differ in certain experimental conditions,
raising
the question of whether they are suitable for circadian experiments.
Because
the number of oscillating genes is tissue dependent and because there
is no
guarantee that a gene oscillating in one tissue will also oscillate in
the
other, and vice versa , we tested several possible reference
genes to
determine the ones best suited for normalization. A large scale
circadian experiment
involving 108 wild-type and 96 Crem knock-out mice has been
conducted.
Mice of similar age were housed with free access to food and water in a
light-dark cycle (light: 7:00am to 7:00pm) for 14 days before they were
sacrificed in dim red light every 4 h in a 24 h period. Liver samples
were
quickly excised, frozen in liquid-N2 and RNA was isolated from 54 wild
type and
45 knock-out male mice using TRI-reagent (SIGMA). In the initial
experiment
cDNA from seven time points was synthesized from 1 µg of RNA and
qRT-PCR
reaction efficiency with serial dilutions was determined for four
genes, two
proposed reference genes (cyclophilin and ß-actin) and two genes
under
circadian control ( Cyp51 and Npas2 ). The aim was to
evaluate
cDNA quality and to determine the cDNA dilution to be used for
subsequent
high-throughput qRT-PCR. The
expression profiles of the four
genes were as expected. Npas2 and Cyp51 showed
oscillation, while
cyclophilin and ß-actin were fairly constant, although there was
a
non-significant difference between some time points. Whether this is
statistically significant needs to be proven with a larger sample
number which
is currently underway. The subsequent study will include the expression
profile
of all 99 samples from male mice for at least 5 possible reference
genes and 2
known circadian genes. The application of three different selection
programs
(geNorm, BestKeeper and NormFinder) will aid in assessing the adequacy
of
selected genes to be used for normalization of the mouse liver
circadian
experiments. P032 Housekeeping
genes validation in acute and chronic adjuvant arthritic rat for mRNA
quantification
by real time RT-PCR Karolinska
Institute, Real time
RT-PCR is one of the
efficient methods in mRNA quantification. One of the approaches for
quantification is the normalization of mRNA by internal control gene or
housekeeping gene. The expression level of internal control should
remain
constant in different tissue types and in different experimental
conditions for
efficient quantification. In the present study we have compared the
expression
levels of 10 commonly used housekeeping genes in 9 different tissue
types in
normal, acute and chronic adjuvant arthritic conditions in rat. We have
also
studied CD3 gene expression in ankle joint and cortical bone to
evaluate the
differences in the levels of housekeeping gene expressions due to
inflammatory
cells. Our data showed an increased of all HKGs in acute and chronic
adjuvant
arthritis. HPRT was the most stable housekeeping gene among all the
tested ones
in acute and chronic adjuvant arthritic conditions in most of the
tissue types
in rat except for the ankle joint and cortical bone. There was no
stable HKG in
both the tissues in acute and chronic condition. We have also found a
significant increase in CD3 gene expression which was significantly
correlated
with the increased expression level in ankle joint and cortical bone in
both
acute and chronic adjuvant arthritic rats. In conclusion our data
suggested
HPRT to be the most stable housekeeping gene in most of the tissue
types in
adjuvant arthritic condition in rat. Further more an increase in the
level of
housekeeping gene expression in ankle joint and cortical bone is due to
the
increase of inflammatory signatures in acute as well as in chronic
adjuvant
arthritic rats P033 Comparison of high and low virulence serotypes of Actinobacillus pleuropneumoniae by quantitative real-time PCRDanish
Veterinary Institute,
Technical University of Denmark, Denmark; kksc@vet.dtu.dk Until now, 15
different serotypes of Actinobacillus pleuropneumoniae (Ap)
have been described
based upon
differences in the capsular polysaccharides of the bacterium. The
virulence of
different serotypes of Ap has been experimentally determined and the
differences in mortality and morbidity are considerable. The genetic
mechanisms
behind these variations in virulence are largely unknown, and for
bacteria in
general, the non-virulent strains often contain many of the virulence
genes
required for an infection. In P034 The
effect of nucleic acid extracts on the reverse transcription step 1Centre
of Infectious Diseases and International Health, University College
London,
United Kingdom; 2University College London Hospitals NHS
Trust,
London; 3Heart Hospital, University College London
Hospitals NHS
Trust, London; g.rodger@ucl.ac.uk
Reverse
transcription quantitative
PCR (RT-qPCR) is an established and accurate method for measuring RNA
from
clinical samples. The reverse transcription (RT) is described as being
the most
variable step of RT-qPCR analysis, yet can be overlooked when
considering
optimisation and quality control. The effect of sample inhibition upon
the qPCR
step is well recognised, with the risk of producing underestimated
measurements
or even false negative results. However, there is little information or
discussion regarding the same effect on the RT step. In this study we
specifically investigate potential inhibition of the RT reaction by
examining
the impact of clinical nucleic acid extracts on the result. In a model
of
infective endocarditis RT reactions were performed on bacterial RNA in
the
presence of different concentrations of nucleic acid extract from human
heart
valve material. The effect of the heart valve extracts on both the RT
and PCR
steps were then assessed by comparing to an uninhibited control
reaction. Our findings
demonstrate that human
heart valve extracts can considerably inhibit the RT reaction. While
samples
that inhibited the RT step were also more likely to inhibit the qPCR
step the
former was automatically at more risk due to the stepwise nature of the
protocol (i.e. the extract always being more concentrated in the RT
than the
subsequent qPCR). Furthermore we demonstrate that some extracts enhance
the RT
reaction. It is clear from our findings that components co-purified
during the
RNA extraction can have considerable positive or negative effects on
the RT
step. This has implications for all RT steps and researchers should be
aware of
the potential important inaccuracies that may occur due to this
previously
undescribed phenomenon. P035 Comparison
of two available platforms for the determination of RNA quality Technical
University Munich,
Germany; irmgard.riedmaier@wzw.tum.de
The integrity
of RNA is a very
critical aspect regarding downstream RNA based analysis. Low-quality
RNA can
compromise the results of such experiments. To save time, costs and
material,
platforms for the determination of RNA quality play an important role.
Two
available systems for the determination of RNA quality are the Experion
(Bio-Rad) and the Bioanalyzer 2100 (Agilent Technologies). Both
platforms determine
RNA quality either by using the ribosomal 28S /18S ratio, or a
numerical system
which represents the integrity of RNA. The Bioanalyzer offers the RIN
algorithm
(RNA Integrity Number) on the Bioanalyzer 2100 and Bio-Rad developed a
new
Experion software version that offers an algorithm for calculating the
RNA
Quality Index (RQI). The aim of these experiments was to compare both
systems
regarding sensitivity, reproducibility and the influence of individual
tissue
extractions and different runs. Therefore RNA quality of 6 different
bovine
tissues was determined in six extraction replicates. Moreover the
connection
between measurement sensitivity and RNA quality should be shown by 6-11
dilution step series with artificially degraded RNA. Every chip
composition was
simultaneously measured in the Experion and the Bioanalyzer 2100. The
experiments show that results obtained from both systems are
significantly
different between the extractions applied, but not influenced by
multiple runs
and chips. Regarding reproducibility and absolute sensitivity, it could
be
demonstrated, that data obtained by measurements on the Experion show
slightly
better results. In the lower RNA quality areas (RIN/RQI 3 to 5) the
Bioanalyzer
2100 shows a higher linearity. Overall it was shown that both
algorithms are
very comparable and beneficial for the determination of RNA quality for
downstream applications, like qRT-PCR or hybridization arrays. P036 Evaluation
of different extraction protocols of total RNA and microRNA from bovine
blood
for gene expression analysis Technische Blood is the
most important tissue
source for molecular diagnostics because it is most easily accessible.
Quantitative real-time RT-PCR (qRt-PCR) has improved the efficiency of
gene
expression analysis. But sampling, handling and the extraction
procedure can
highly impact variability of gene expression results. This led us to
compare
five different blood RNA sampling and extraction protocols to find out
which
guarantee the best outcome with respect to highest yield of RNA and
microRNA concentration
and lowest crossing point (CP) with least variance in qRT-PCR
applications. Blood
samples were collected in triplicate from 5 healthy Brown Swiss
heifers. Two
phenol based extraction methods [Trifast], namely direct extraction of
whole
blood (WB) and leucocyte extraction after lysis of erythrocytes (LY)
were
designed to isolate total RNA including mRNA and microRNA in one
fraction.
PAXgene [Qiagen] (PAX) and LeukoLOCK [Applied Biosystems] (LL) sampling
devices
as well as the extraction of the plasma interphase of coagulated
centrifuged
blood (PI) were performed to separately isolate mRNA (>200nt) and
small RNA
(<200nt) [Qiagen]. RNA and microRNA were extracted from either whole
blood
(WB and PAX) or blood leukocytes (LY, LL and PI). For each extraction
protocol
RNA yield was quantified [Nanodrop], quality and integrity was obtained
[BioAnalyzer] and gene expression was measured by qRT-PCR [Eppendorf]
of 11
mRNA and 7 microRNA target genes. The best results in terms of highest
quantity, quality and best performance for mRNA qRT-PCR were obtained
for the
samples extracted by the LY and PAX methods. Moreover there is a
tendency to
obtain the best microRNA results for the samples where total RNA was
extracted
in one fraction (LY and WB). The PI method show the worst results due
to high
variance in all variables considered in this study. Since normalization
could
reduce the effect of RNA integrity on gene expression the extraction of
whole
blood or leucocytes showed minor differences. In summary, the results
obtained
offer an overview of practical concerns of different mRNA and microRNA
extraction methods from bovine blood. As superior quality parameters
where
obtained with different extraction protocols, the method of choice has
to be
based on the necessity of the study conditions. P037 Lek
Pharmaceuticals d.d., a Sandoz
company, Verovškova 57, SI-1526 Ljubljana, Slovenia; mihaela-1.skulj@sandoz.com
The majority
of human therapeutic
proteins are favorable to be produced in mammalian cell-culture systems
due to
their ability of proper folding, postranslatic modifications and
glycosylation
profiles of therapeutic proteins. Since determination of transgene copy
number
and genetic stability are among the important parameters for
recombinant
protein production, the focus of this study was the application of qPCR
method
for this purpose. In presented work Chinese hamster ovary (CHO) dhfr-
cell line
was used for expression of the recombinant protein underwent antibiotic
and
methotrexate selection. A method for gene copy number determination
using
absolute quantification and TaqMan chemistry was developed. Specific
primers
and probes for transgenes of A and B subunit of recombinant protein and
endogenous gene on CHO chromosomal DNA were constructed. A ratio of
copy number
between recombinant and endogenous gene was calculated and presented as
a
transgene copy number per diploid genome of CHO cell. The method was
successfully
applied for genetic characterization and stability study during
development and
final characterization of cell line for recombinant protein production.
In
addition some interesting findings were observed, a correlation between
gene
copy number and specific productivity after amplification with
methotrexate as
well as a correlation between the amount of plasmids containing
recombinant and
resistance gene respectively, used for cotransfection in comparison to
the
transgene copy number. P038 Comparison
of two different types of preamplification 1Laboratory
of Gene ExpressionInstitute of Biotechnology, Academy of Science, Czech
Republic; 2Department of Tumor Biology3rd Medicine Faculty,
Charles
University, Czech Republic; vendula.rusnakova@img.cas.cz
Accurate gene
expression
quantification by qRT-PCR could be limited by a low RNA quantity
obtained from
clinical samples (laser microdissection, circulating tumor cells or
FFPE tumor
tissue). One of the method that helps to overcome the small amount of
the
sample material and to lower the originally high qRT-PCR CTs is
preamplification of cDNA. Here, we present the comparison of two types
of
preamplification, preamplification of cDNA using the TransPlex Whole
Trascriptome Amplification Kit and gene specific preamplification. We
compared
expression profile of 40 genes (breast cancer markers and housekeeping
genes )
in FPPE tumor tissue. We used Biomark instrumentation for qRT-PCR. The
molecular profile of the samples was analyzed by biostatistical method. P039 Effects
of formalin, methacarnoy and FineFIX fixation on RNA recovery and
preservation 1University
of Archival
formalin-fixed and paraffin
embedded tissues represent the most abundant supply of clinical
material on
which molecular analyses can be now performed for translational
studies.
Formalin, however, has detrimental effects on nucleic acids and
proteins, impairing
their extraction efficiency and suitability. Moreover, formalin
represents a
hazardous chemical component. In recent years several alternative
fixatives
have been introduced in order to overcome these limitations. In the
present work, formalin was compared
with two alcohol-based fixatives, methacarnoy and the non toxic
commercial
FineFIX, with respect to their effect on RNA recovery and preservation.
The
effect of time of fixation (from 1 to 24 hours) was evaluated in a cell
line-based model. The effects of fixatives were then investigated in a
panel of
human tissue samples to evaluate the impact of further variables (e.g.
time of
fixative penetration, tissue composition) on RNA impairment. Fresh
cells and
tissues were used as control. Total RNA yield was measured by Nanodrop,
rRNA
integrity was analysed by Agilent Bioanalyzer, mRNA integrity by
endpoint
RT-PCR amplification of fragments of increasing length and mRNA
quantity by
realtime RT-PCR. In the cell
line model RNA recovery
as well as rRNA and mRNA preservation were affected by all the fixation
procedures, although to a different extent. Formalin fixation showed a
time-dependent detrimental effect on both total RNA recovery, rRNA/mRNA
integrity and mRNA quantity. Methacarnoy and FineFIX resulted more
conservative
than formalin on RNA yield, mRNA integrity and quantity and their
effect was
time-independent. However, RNA from methacarnoy-fixed samples showed a
better
rRNA preservation with respect to that from FineFIX-fixed specimens. In
tissues, high rRNA degradation levels were noticed in all the
differently fixed
specimens, while the detrimental effects of formalin and the protective
effect
of both alcohol-based fixatives on mRNA integrity were all confirmed.
Accordingly, in methacarnoy-fixed samples also mRNA quantity was
preserved,
resulting comparable to that obtained in controls. Conversely, in
FineFIX-fixed
samples mRNA expression results were quantitatively similar to those
obtained
with formalin treatment. In
conclusion, we showed that both aldehyde-
and alcohol-based fixatives have detrimental effects on overall RNA
preservation but the entity of their effects is fixative-specific and
is
dependent on variables related to the use of tissue samples. In
particular, the
time-independent effects of alcohol-based fixatives make them practical
for RNA
investigations even in specimens fixed for very long periods. Moreover,
in our
hands methacarnoy results the most suitable fixative for RNA analyses
in small
clinical samples because it ensures the highest level of RNA recovery
and
preservation. P040 Real
time monitoring of cell behavior on the xCELLigence system supports
qPCR
analysis of dynamic gene expression Application
Lab, Roche Applied
Science, The analysis
of gene expression
patterns after candidate drug treatment of cell lines is a crucial step
in
understanding drug activity and in the identification of biomarkers
which
supports the identification of patients who will benefit from a new
drug. Here
we describe a model system in which we combine the online monitoring of
cellular events after drug treatment using the xCELLigence system with
qPCR
analyses using RealTime ready panels. Cell growth curves recorded by
the
xCELLigence system after treatment with candidate drugs for the first
time
provide means to identify optimal time points for sample collection for
subsequent RT-qPCR analysis. Here we treated the colon cancer cell line
HT29
with a previously defined effective concentration of the anti-cancer
drug
paclitaxel. The growth behavior of the treated cells was monitored in
comparison to the growth behavior of control cells. The expression
level of 84 apoptosis-related
genes was compared among treated and untreated samples collected at
different
time points using the Real Time ready Human Apoptosis Panel, 96 on
LightCycler®480 Instrument. For data analysis the
LightCycler®480 Multiple
Plate Analysis Software was used. The results show that xCELLigence
supports
the knowledge based decision for ideal time points in the analysis of
dynamic
gene expression patterns by RT-qPCR. P041 STABILITY
OF ENDOGENOUS CONTROL GENES IN HUMAN POST-MORTEM TISSUE 1University
of The
possibility to use human tissue
obtained during forensic autopsy for extraction of RNA in suitable
quantity and
quality for gene expression analysis was shown previously. Nevertheless
it is
undoubted that special care should be taken when working with human
tissue samples
from individuals with long postmortem intervals which are common in
forensic
routine work. A correct data normalisation strategy that is able to
eliminate
effects from changing postmortem conditions as well as various causes
of death
is indispensable before performing gene expression studies. In our study
we examined the
stability of 10 potential endogenous control genes in different human
postmortem tissues by calculating an average expression stability
value. The
stability of these genes will be presented with respect to different
causes of
death and varying postmortem intervals. The minimal number of
endogenous
control genes needed for correct data normalisation in gene expression
studies
in human postmortem tissue as well as appropriate candidate control
genes will
be presented. P042 SYTO9-based
real-time PCR and electron microscopy for quantification of colloidal
gold-bound oligonucleotides and antibodies Colloidal
gold nanoparticles (GNPs)
armed with oligonucleotides alone or together with antibodies are
increasingly
used for sensitive detection of DNA or proteins. An important step for
optimizing the sensitivity and reproducibility of GNP-based assays is
the
quantification of oligonucleotides bound to the particles. To this end,
several
techniques including real-time PCR with fluorescent beacon probes have
been described.
However, these techniques are either insensitive or require expensive
probes.
Here we report a SYTO9-based real-time PCR method for quantification of
oligonucleotides bound to GNPs. Thiol-capped oligonucleotides were
attached to
30 nm GNPs (30GNPs). Unbound oligonucleotides were removed by
centrifugation of
the particles through 30% glycerol. Pelleted particles were then
dispersed in
phosphate buffer (pH 7.4) with 0.15 M NaCl and 1% bovine serum albumin
(BSA).
In some experiments, rabbit polyclonal antibody specific for selected
antigen
was bound to 30GNPs, followed by binding of thiol-capped
oligonucleotides. To
detect gold-bound oligonucleotides, the particles were pelleted and
resuspended
directly in 1x PCR master mix containing SYTO9 fluorescent dye, TaqDNA
polymerase, dNTPs, buffer, short template DNA complementary to the 3'
end of
the oligonucleotide bound to 30GNPs, and two unlabeled primers
complementary to
the 5´ end of the gold-bound oligonucleotide and 3' end of the
template.
Routinely, 40 cycles of PCR were monitored using real-time Mastercycler
Realplex (Eppendorf). No inhibitory effect of 30GNPs was detected on
SYTO9-based real-time PCR. An indirect relationship was evident between
the
amount of oligonucleotides bound to 30GNPs and the Ct values obtained.
Calibration curves were constructed using free thiol-capped
oligonucleotides.
Rabbit antibodies bound to 30GNPs were detected with commercial 5 nm
GNPs armed
with goat-anti rabbit IgG (GaR_5GNPs). Briefly, armed 30GNPs were bound
to an electron
microscopy grid covered with pioloform film and poly-L-lysine. After
washing
and blocking with BSA, GAR_5NPs were added. Unbound particles were
removed by
washing and dry samples were observed by electron microscope. When
properly
prepared, 30GNPs with bound rabbit antibodies were surrounded by
GaR_5GNPs
forming rosette-like structures. Usually, >90% antibody-armed 30GNPs
formed
rosettes. When 30GNPs were armed with unrelated protein, almost no
GAR_5GNPs
were bound, confirming the specificity of the assay. The results
indicate that
SYTO9-based real-time PCR offers a rapid, simple and inexpensive method
for
mapping the surface coverage of oligonucleotides bound to GNPs. In
combination
with detection of antibodies bound to the same particles it is possible
to
monitor properties of nanogold conjugates. Such data are extremely
important in
standardization of bio-barcode amplifications and other assays used for
detection of proteins. Supported by KAN200520701. P043 The
tilted threshold line. An alternative to the horizontal counterpart
which is
commonly used for dilution curve quantification? University
Hospital The
estimation of real-time
efficiencies has been shown to be conducted most accurately by
employing
calibration curve analysis. For this purpose, serial dilutions of the
gene of
interest are subjected to real-time PCR and the threshold cycles
obtained from
the dilutions are plotted against the logarithmized dilution factors
(relative
approach) or copy numbers (absolute approach). The threshold cycles (or
otherwise denoted as crossing points) for the above procedure are
usually
acquired from the software accompanying the real-time PCR instrument.
This
value is instrument specific and may often be not be very intuitive for
the
user. A major
drawback is to our opinion,
that the threshold cycles are always calculated from a fluorescence
value which
is constant over all dilutions and samples (threshold line or border).
This
results in suboptimal regression curves when the difference in the
threshold
cycles (delta-ct’s) between two dilutions gets larger with increasing
dilutions, which is almost always seen for higher cycles. Being able to
accurately quantify samples with very low abundance of transcripts is
crucial,
as these often belong to the most interesting biological groups (i.e.
transcription factors or receptors). We developed
an approach which might
tackle this problem. Our method subtitutes the horizontal threshold
line by a a
line which is tilted downwards from the sample of lowest dilution to
the sample
of highest dilution. This line with slope X and intercept Y is
calculated
iteratively through all intercepts and slopes (calculation time about
2-10
minutes, depending on the number of steps chosen within each
iteration), and
ultimately a threshold line with defined X and Y is selected that
optimizes the
performance of a regression curve, either by maximizing the coefficient
of
determination R-square or by minimizing the Akaike Information
Criterion (AIC).
Constraints are applied to this curve such that the iteration starts at
the
second derivative maximum of the lowest dilution sample and stops at
the first
outlier cycle of the highest dilution curve, so that values in the
baseline
region of the curves are not accidentally included. We show that with
this
method, a gain in accuracy and precision for those regimes can be
achieved,
where there is a need in quantitation of very low abundance genes. P044 Establishment
of robust normalizers for the qRT-PCR analysis of kidney and heart
under
diabetic conditions and upon the treatment with the copper(II) chelator
TETA in
R. norvegicus 1School
of Biological Sciences, Objective:
Diabetic nephropathy and
diabetic cardiomyopathy are severe diabetic complications. An
experimental
treatment for the diabetic cardiomyopathy and the diabetic nephropathy
is a
normalization of the copper homeostasis in these organs using the
Cu-selective
transition metal chelator trientine (TETA) (Cooper et al .,
2004). We
aim to better understand the molecular mechanisms by which TETA
treatment leads
to the reverse of diabetic heart and kidney function in the animal
model of
STZ-induced diabetic rats. Here, we report the establishment of robust
normalizers suitable for qRT-PCR assays of diabetic heart and kidney
and show
the importance of proper normalization in the context of the expression
analysis of genes of glutathione metabolism. Methodology:
Animal experiments
using STZ-rats were performed according to ethical standards. Total RNA
was
extracted from left ventricle of hearts and renal cortex, followed by
cDNA
synthesis. cDNA concentrations were determined and equal amounts of
cDNA were
used in each qPCR run. Standard and melting curves were performed for
every
gene in every run. An efficiency corrected ΔCt method was used for
calculation
of relative expression levels. Candidate reference genes were analyzed
via the
NormFinder (Andersen et al ., 2004) and the geNorm
(Vandesompele et
al ., 2002) algorithms. The relative expression levels of our
genes of
interest were calculated according to Pfaffl et al . (2004). Results: We
analyzed U2AF, NDC1,
RPL13a, PIPA, YWHAZ, TBP, GAPDH, bACT and 18S as candidate reference
genes.
Total cDNA was also analyzed as a possible normalizer. U2AF, TBP and
bACT were
rated the best candidate reference genes in kidney according to both
algorithms. For the heart both algorithms rated NDC1, TBP and RPL13a as
the most
stably expressed candidate reference genes. For the
expression of Glutathione
S-transferase kappa 1 we show that usage of a ‘bad’ normalizer can
reverse the
trend observed with a proper normalizer. Conclusions:
We have identified sets
of reference genes to build robust normalizers for qRT-PCR analysis of
kidney
and heart under diabetic conditions and upon the treatment with TETA.
We
further show for the first time that U2AF and NDC1 are reasonable
candidate
reference genes, expanding normalization to genes coding for
spliceosomal
components and parts of the nuclear pore complex. Abbreviations:
TBP: TATA-box binding
protein, NDC1: Nucleoporin1, U2AF: U2 snRNP auxiliary factor, bACT:
β-Actin,
GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, PIPA: Peptidylprolyl
isomerase
A, YWHAZ: Tyrosine 3-monooxygenase, 18S: 18S rRNA, RPL13a: Ribosomal
protein L
13a References:
Cooper et al. (2004)
Diabetes 53:2501-2508, Andersen et al. (2004) Cancer Research
64(15):5245-50,
Vandesompele et al. (2002) Genome Biology 3: 0034.1-0034.11, Pfaffl, et
al.
(2004) Biotechnol Lett 26(6):509-515 P045 Lek
Pharmaceuticals d.d., a Sandoz
company, Verovškova 57, SI-1526 Ljubljana, Slovenia; veronika.okrslar@sandoz.com
In
biopharmaceutical industry
stringent requirements take place for validation of analytical methods
used for
characterisation of products and cell lines producing them. Genetic
characterization of cell lines chosen for production, from which MCB
(master
cell bank) and WCB (working cell bank) are prepared, is a first step
under the
cover of GMP in a process of recombinant protein production
development. In
addition to prove sequence by sequencing, control length and
differentiate
integration sites of transgene by Southern blot analysis and check
length of
transcript by Northern blot analysis, a qPCR method for transgene copy
number
determination was introduced. For this purpose the qPCR based method
had to be
validated in a proper way according to the guidelines. Particular
assays were
designed to prove the accuracy, precision (intra-assay, intermediate),
specificity, selectivity, linearity, determine limit of detection,
limit of
quantification, range of quantification and test ruggedness and
robustness of
the developed method. On a basis of data collection during method
qualification
acceptance criteria for above listed assays were set. In this work,
case study,
challenges, difficulties not easy to overcome and solutions arisen
during the
design of assays and testing of parameters are addressed and presented.
The
outcome of our work is validation protocol, which is used in our
laboratory to
verify qPCR method for transgene copy number determination and assure
reliability of the obtained results. P046 Roche
Diagnostics GmbH, Roche
Applied Science, With the new
introduced RealTime
ready Focus Panels, Roche Applied Science now offers a convenient and
reliable
solution of content supply specifically for the LightCycler® 480
System. The
RealTime ready assays are based on the unique Universal ProbeLibrary
technology
which allows fast and flexible assay design for millions of targets
from
virtually any organism for Gene Expression Profiling applications. The
Universal ProbeLibrary consist of just 165 short probes providing
transcriptome-wide coverage in most organisms. These short probes are
highly
specific and possess the high melting temperature (Tm) required for
real-time
PCR due to the incorporation of locked nucleic acid (LNA). Each RealTime
ready qPCR assay is
extensively validated covering sensitivity, PCR efficiency, specificity
and
signal intensity. The RealTime ready assays are dried-down in micro
well plates
and currently available as content panels (specific for certain
cellular
pathways, protein or gene families etc). The relevant genes for certain
pathways and gene families were selected in cooperation with experts
and are
now available as pre-plated, ready-to-use assays. Full
customization of RealTime ready
is planned for 2009. This will enable researchers to create their own
panels on
an easy-to-use online configuration portal or to order single assays
for their
targets of choice. The presented
results demonstrate
that the RealTime ready Focus panels show very high performance in
real-time
PCR based expression assays compared to other technologies. These new
tools
offer major advantages like the combination of high flexibility and
high
specificity and additionally a reduction in time from assay design to
experiment. Thus, the RealTime ready Focus panels provide a robust
expression
profiling platform highly useful for high-throughput expression
analysis,
especially for microarray validation and gene knock-down experiments. P047 Mechanism
of mRNA degradation in Xenopus laevis Institute of
Biotechnology AS CR,
Videnska 1083, Cell
determination during early
development depends on mRNA and protein expression and the distribution
of the
molecules within and among cells. Also mRNA degradation plays an
important role
in early development. Production and degradation of mRNAs must be
precisely
controlled for normal development of an organism. Our aim is to study
mRNA
degradation mechanisms during early development of the African clawed
frog
Xenopus laevis that occur post mortem. We applied real-time RT-PCR and
a new 5’
/ 3’ approach. Our results suggest that post mortem mRNA degradation is
rate
limited by the initiation of the degradation process. Once this starts
the
entire mRNA is rapidly degraded. This work is
supported by GAČR
301-09-1752 and GAAV IA500970904 P048 Easy
and Reliable qRT-PCR Analysis of Total RNA Isolated from Fresh Frozen
and FFPE
Tissue Samples Application
Lab, Roche Applied
Science, The use of
tissue sections for
molecular analysis of pathogenic states in mammalian tissue has become
an
indispensable approach for the understanding of molecular mechanism in
etiology
and disease progression. In particular, tumor genesis, as a very local
process,
requires individual sample preparation and conservation. Most commonly,
dissected tissue samples are prepared as fresh frozen or even more
often as
Formalin-Fixed Paraffin Embedded (FFPE) samples. Both techniques have
advantages and disadvantages. The most severe drawback of FFPE samples
is the
degradation of RNA during the sample conservation. Although fresh
frozen sample
material better reflects the pathological state of a tissue, the
challenge to
economically organize the required logistical chain - necessary for
fresh
frozen tissue samples - have not been satisfactorily solved. Here we
present
data for two independent workflows for gene expression analysis using
Roche’s
LightCycler®480 Instrument with the SYBR Green I and the UPL
format. RNA
samples were isolated from FFPE or fresh frozen HeLa xenograft tissue.
Either
the manual method using the High Pure FFPE RNA Micro Kit or the
automated
method using the MagNA Pure LC RNA Isolation Kit III (Tissue) on the
MPLC 2.0
Instrument was employed. Isolated RNA was subjected to cDNA synthesis
using the
Transcriptor First Strand cDNA Synthesis Kit. The results show that a
robust
workflow leading to excellent data sets suitable for gene expression
analysis
can be established by combining Roche products. P049 Bio-Rad
Laboratories, Inc. 2000 Reducing run
times for PCR and
real-time PCR assays can dramatically increase throughout and
accelerate
scientific discovery. Next generation instruments and reagents open the
door to
generating rapid and robust assays, but steps must be taken to ensure
compressing run times is not detrimental to the sensitivity and to the
reproducibility of low expression targets. With Bio-Rad’s next
generation of
real-time PCR supermixes, fast real-time PCR can be achieved without
sacrificing assay reproducibility or sensitivity. We present strategies
and
techniques to reduce PCR and real-time PCR assay times without
compromising the
quality of results. microRNA – siRNA
Applications
P050 Molecular
phenotyping of miRNA perturbation in neuroblastoma 1Applied miRNAs are an
emerging class of
small non-coding RNAs involved in the regulation of oncogenes, tumor
suppressors, and a number of cancer-related genes. The potential of
miRNAs as
therapeutic molecules and targets in cancer pathogenesis has increased
the
urgency of studying miRNAs in the field of cancer research. This
abstract
presents an innovative study, in which functional screening of both the
whole
human Pre-miR™ and Anti-miR™ libraries is performed on different
neuroblastoma
cell lines, followed by the expression profiling of various genes
involved in
cancer-related pathways. Neuroblastoma
(NB) is the most
common extracranial solid tumor of childhood and comprises of about 15%
of all
childhood cancer deaths. The whole human miRNA assay panel was first
used to
profile miRNAs involved in MYCN-amplified neuroblastomas. Several miRNA
were
shown to be fundamental components of the MYCN transcriptional network
that
mediate the MYCN tumorigenic program. Transfection of NB cell lines
with miRNA
inhibitors and precursors directed against the up- and down-regulated
miRNAs
respectively resulted in a profound decrease of cell viability
indicative for
oncogenic and tumour suppressor activity. Functional
screening of 470 Pre-miR™
and 470 Anti-miR™ will be performed next on 10 neuroblastoma cell
lines. Some
of these cell lines are used as control, others have interesting
aberrations in
genes known to play a role as oncogene or tumor suppressor (such as
MYCN
amplification). After transfection, the cells are lysed with
Cells-to-Ct and
expression profiling is done by RT-qPCR on a panel of 20 genes involved
in cell
cycle, apoptosis, cell migration, and angiogenesis. The first
findings indicate that
miRNAs are fundamental components of the MYCN tumorigenic program. The
whole
Anti-miR™ and Pre-miR™ library screening is an innovative way to
discover which
miRNAs are involved in the regulation of important pathways related to
cancer
pathogenesis and thus potential new therapeutic molecules in cancer
treatment. P051 Isolation
of small RNA species from PAXgene Blood Tubes QIAGEN, Sampling of
blood is a standard
procedure in most clinical trials and many diagnostic testing
procedures.
Pathological conditions in organs and remote tissues are often
detectable in
gene expression profiles from blood samples. Artificial modifications
of the
RNA content and profile of a given blood sample between collection and
isolation procedure caused by degradation and gene induction are well
documented (Rainen et al. 2002 Clin Chem 48: 1883-90; Müller et
al. 2002
Leukemia 16: 2395- 99). Doubtful results are the consequence especially
for
very sensitive analysis methods like qRT-PCR assays and gene chip
experiments.
So the need for stabilisation of cellular RNA species to freeze the
gene
expression profile at the time of blood collection is widely accepted
and the
PAXgene Blood RNA stabilisation and isolation systems are commonly used
to
address this problem. Since the
standard PAXgene Blood RNA
protocols were designed for mRNA purification the isolation of miRNA
from
PAXgene Blood RNA tubes is not very efficient, as the binding
conditions are
not optimized for small RNA species. A first attempt to optimize these
conditions and to allow a more efficient purification of small RNAs
from
PAXgene Blood RNA tubes was published recently (Kruhøffer et al.
2007 Vol 4(4):
452-8). Due to several drawbacks of this method, we developed a new
protocol
and chemistry to achieve optimal yields of small RNAs from PAXgene
Blood RNA
Tubes. Yield and
quality of small RNA species
were determined on the Nano and small RNA Labchip using an Agilent®
2100
bioanalyzer as well as with classical gel analysis. Purified RNA was
analyzed
for genomic DNA contamination and the presence of PCR inhibitors. As a
downstream method different quantitative RT-PCR assays for miRNAs based
on SYBR
green or probes were used. We could show
a clear enrichment of
small RNAs by gel analysis and on different Agilent Bioanalyser chips
for
manual as well as for automated miRNA protocols compared to the
corresponding
standard PAXgene protocols. High miRNA contents could be confirmed with
different qRT-PCR assays detecting miR-10a, 16, 30b, 192 and let7a.
Very low
amounts of gDNA (< 1%) were present in the eluates and they showed
no
inhibition of RT-PCR. We saw no interference with qRT-PCRs when
detecting
different mRNA transcripts. These results
show that the
dedicated isolation procedures for small RNAs based on the PAXgene
Blood RNA
Tube result in high quality enrichment of these RNA species which are
ready for
direct use in sensitive downstream applications. Disclaimer:
PAXgene Blood miRNA
system is intended for Research Use Only. Not for use in diagnostic
procedures. P052 Quality
control of miRNA in biological extractions Agilent
Technologies, In recent
years, hundreds of miRNAs
have been discovered, but their functional roles remain to be
classified in
detail. The expression level of individual miRNAs is typically
quantified by
methods like qRT-PCR or microarray hybridization. One of the major
drawbacks in
miRNA research is the lack of adequate analytical methods for small RNA
extractions before going in the subsequent analysis of miRNA fractions. Here we
present a highly sensitive
microfluidic assay for the analysis of small nucleic acids. The assay
allows
detecting miRNA fraction down to a concentration of 50 pg. It measures
integrity, size and concentration of small RNA and is especially
calibrated for
miRNA. Those features allow researchers to optimize small RNA isolation
and
purification protocols, to monitor the ratio of miRNA fractions in
total RNA
extracts, and to screen siRNA preparations. In conclusion, our approach
allows
the implementation of miRNA quality control in an expression workflow
by
monitoring miRNA fractions derived from different tissues or extraction
methods. P053 Gene
expression profiling of 56
colorectal cell lines by qRT-PCR was performed against a panel of 384
miRNAs
using Low Taqman Density Arrays (TLDAs, Applied Biosystems). Cell lines
were
predominantly isolated from patients with Dukes stage B-D disease and
there are
two lines from stage A (SW1116 & C106). Data analyses of drug
sensitivity
for the colorectal cell lines have identified: 13 significantly,
differentially
expressed miRNAs common to two oncology compounds that are EGFR
inhibitors and
10 miRNAs significantly, differentially expressed miRNAs common to two
oncology
compounds that are MEK inhibitors. These miRNAs are potentially
predictive of
drug sensitivity in in vitro models of colorectal cancer. Additionally,
the linkage between
mutation status for BRAF, KRAS and p53 and miRNA gene expression of the
colorectal cell lines was tested. Statistical analyses were performed
on a
subset of 177 miRNAs for the following groups: BRAF (37 wild type &
10
mutant), KRAS (20 wild type & 25 mutation) and p53 (10 wild type
& 34
mutation). Permutation testing revealed that the most significant miRNA
gene
expression change was with p53 mutation status. 20 miRNAs with highly
significant, differential gene expression (p < 0.05) have been
identified
and correlated to p53 mutation status. Significant differential miRNA
gene
expression linkage for BRAF and KRAS in this colorectal cell line panel
was not
demonstrated. This study
has validated the
potential of miRNA gene expression as a powerful tool to identify
biomarkers
for the prediction of drug sensitivity and linkage to mutation status
in
colorectal cell models. P054 qPCR
applications for Drug Discovery In the CVGI
(Cardiovascular and
Gastro-intestinal) department at Astrazeneca our work focuses primarily
on
diabetes and obesity research. We routinely use the QPCR technique
throughout
all stages of the drug discovery process from target validation through
to lead
optimisation, for tissue expression profiling, assessing the effects of
treatments and diet on gene expression, verifying knockdown of a gene
by siRNA
and assessing expression of a transgene in transgenic mice. We run our
qPCR on
the ABI 7900 platform, which allows us to utilise 96 well, 384 well and
microfluidic card formats. For larger studies we use an automated
system from
Qiagen, the Qiacube, to prepare RNA from our homogenised samples and
are
currently looking into automated systems to optimise the other steps in
the
process. P055 Next
generation siRNAs: combining innovative design with novel chemical
modifications, for superior consistency of phenotypic results. Applied
Biosystems; emmanuel.louis@eur.appliedbiosystems.com
Poor efficacy
of siRNA prediction
and poor specificity too often combine to wreak havoc for RNAi users.
To
alleviate these problems, we used classification bioinformatics and
novel
nucleic acid chemistry to produce the next generation of siRNA
technology
resulting in improved coherence of phenotypes between siRNAs to the
same mRNA
and higher siRNA potency. We employed a support vector machines (SVM)
strategy
to predict highest potency siRNA. Maximum silencing was obtained with
90% of
the siRNA predicted using this algorithm at doses 5-100x lower than all
other
currently commercially available siRNAs. In addition we compared
LNA® and 2’OMe
in strategic placements of modifications throughout the siRNA structure
and
found a novel pattern of LNA® placement that improves siRNA
specificity without
disrupting the potency of the siRNA. The top ranking siRNAs predicted
by the
algorithm produce consistent maximum knock-down and the lead
modification gave
the smallest footprint in microarray experiments compared to
alternative
chemistries or unmodified siRNA with the same sequence. High-content
microscopy
testing of a custom library of genes demonstrated that >90% of the
siRNAs
tested elicit the expected phenotype, showed reduced off-target cell
effects
compared to unmodified siRNA and produced coherent phenotypes between
siRNAs.
The SVM strategy, results of the chemical modification screen and RNAi
performance supporting data will presented. P056 The
application of RNAi for studying of isoflavone synthase gene family in
red
clover ILVO, Isoflavones
are mostly restricted to
the Fabaceae family. Within Fabaceae family, red clover has recently
received
considerable interest as a valuable source of isoflavones. It has been
suggested from the previous studies that isoflavones play an important
role in
plant defense system in response to the number of stresses including
attack by
pathogens, UV light, and physical and chemical damage. However, it is
supposed
more concentration of isoflavones increase red clover responses to
biotic and
biotic stress. In this study, RNA interference technique was used to
study role
of IFS gene family, a key gene in isoflavone biosynthesis. Inverted
repeat
binary vector of IFS gene has been made using Gatway technology and
transferred
via Agrobacterium-mediated system to red clover. Using q-RT PCR we
showed
dramatically decrease in IFS gene in comparison to control plants. The
transgenic plants will be investigated by more molecular techniques.
Key words:
qRT-PCR (quantitative real-time polymerase chain reaction), RNA
interference,
Gatway technology, red clover Abbreviations: qRT-PCR, reverse
transcription
followed by quantitative real-time PCR; IFS, isoflavone synthase gene P057 QPCR-based
miRNA Profiling with High Specificity Agilent
Technologies, A miRNA
QPCR-based quantitation
method was developed using polyadenylation to add a polyA tail to miRNA
followed by reverse transcription (RT) and QPCR (Shanfa, L, et al.
2005. Plant
Cell. 17(8): 2186–2203). We have expanded on this method to develop an
assay
that discriminates between miRNAs that differ by as few as a single
nucleotide
as demonstrated by less than 1% cross reactivity between all human
let-7 family
members. The method was validated using 2-250ng input total RNA and
resulted in
a linear signal when quantitating miRNA of varying abundances. A single
polyadenylation and RT reaction allows for profiling of 300-6,000
different
miRNA resulting in extensive miRNA profiling from a very small amount
of total
RNA. The method was used to quantitate miRNA in total RNA from a
variety of
sources including FFPE tissues and in cell lysates. Further validation
of this
method identified differentially expressed miRNA in a matched set of
normal
colon and adenocarcinoma RNA from the same donor. In this study, 50
different
human miRNA were profiled based upon their potential roles in cancer
and
development. This
QPCR-based method allows for
highly specific and sensitive miRNA expression profiling from small
amounts of
RNA to identify miRNA of diagnostic, prognostic, and therapeutic value
in a
variety of diseases. P058 Stability
of microRNA in partly degraded RNA from lung samples National
Veterinary Institute,
Technical University of Denmark, Denmark; kesk@vet.dtu.dk MicroRNA has
gained a lot of
attention recently, due to its involvement in epigenetic regulation of
gene
expression. Expression profiles of these small non-coding RNAs have
also been
used for different diagnostic purposes. The stability of microRNA has
previously been tested in formalin-fixed paraffin-embedded tissue and
has shown
to be relatively high (Li et al., 2007). The small size of microRNA
could be a
possible explanation for a greater tolerance to degradation (Fleige et
al.,
2006). We tested the stability of microRNA in partly degraded RNA
isolated from
lung tissue as little is known about obtaining reasonable qRT-PCR data
of
microRNA expression from partly degraded RNA. Lung tissue from three
healthy
pigs were cut into six pieces of 1cm x 1cm and stored at room
temperature in
Petri dishes in 0 h, 1 h, 8 h, 24 h, 48 h, and 72 h. To the given time
RNA
Later (Invitrogen) were added to stabilise the RNA. Total RNA including
small
RNA was extracted using TRI Regent (Sigma) and the integrity of the RNA
was
determined using two different chips (RNA Nano and Small RNA) on the
Agilent
Bioanalyser. The relative concentration of three well described
reference genes
(B2M, β-actin and GAPDH) was compared with the relative concentration
of three
putative microRNA reference genes: mir-23a, mir-26a and mir-34a. A
correlation
coefficient of 0.87 was found between RNA degradation (RNA integrity
number
(RIN)) and time (h), confirming RNA degradation within the lung tissue
at room
temperature. During the time span of this study RIN decreased from 9.0
(±0.12)
at time 0 h to 4.7 (±1.57) at time 72 h. Electropherograms of
small RNA
confirmed these results, ranging from high quality RNA samples, with a
clear
tRNA peak at time 0 h to highly degraded samples after 72 h. The
concentration
of mir-23a, mir-26a and mir-34a was found to be more or less stable
within the
first 24 h, whereas concentration of the three mRNA reference genes was
found
to decrease within less than 8 h. Previous studies have found RNA
stability to
differ between tissues due to variation in type and quantity of active
ribonucleases as well as differences in tissue structure (Schoor et
al., 2003;
Seear and Sweeney, 2007). In this study we tested the stability of
microRNA and
mRNA in partly degraded RNA isolated from lung tissue. Initial results
indicate
that mir-23a, mir-26a and mir-34a are relatively stable at room
temperature in
lung tissue within the first 24 h. Cancer Centre
and Purpose -
A clear-cut diagnosis of Burkitt's
lymphoma (BL) and its differentiation from diffuse large B-cell
lymphoma
(DLBCL) is of great clinical importance, as therapies for BL and DLBCL
differ
and BL treatment should be promptly initiated. MicroRNAs - short,
non-coding
RNA molecules have been implicated in different pathological processes,
including cancer development and progression, and an aberrant
expression of
numerous microRNAs has been found in multiple human tumor types. The aim of
presented study was to
investigate the potential of selected miRNAs, including miRNA-155, as
biomarkers for the differential diagnosis of BL vs lymphoma DLBCL. Patients, Materials and Methods – Clinical
samples from adult BLs and DLBCLs were characterized regarding
morphologic
features, immunophenotype (flow cytometry) and cytogenetic profiles
according
to the recent lymphoid malignancies WHO classification (2008). All
miRNA
measurements were performed using TaqMan MicroRNA assays
(AppliedBiosystems)
and small nuclear RNA, RNU6 was assayed for normalization. Results - We showed
that the expression of
miR-155 is absent or significantly lower in adult BLs, contrary to
DLBCLs. In
addition, our initial experiments showed a good correlation between the
expressions of miR-155 and, it’s precursor, BIC in BL pathological
samples. The
relationships of the observed microRNA-155 expression pattern with the
clinical
data as well as with the cytogenetic and immunophenotype profiles of
the
analyzed cases will be presented. Conclusion - Our
findings highlight the
importance of microRNA-155 as a potential biomarker in the differential
diagnosis of Burkitt's lymphoma vs diffuse large B-cell lymphoma, and
provide
new insights into the role of miR-155 in the pathogenesis of the
aggressive B
cell lymphomas. P060 MicroRNA
expression profiling in the pig developing brain 1Genetics
and Bioinformatics, Faculty of Life Sciences, Copenhagen University,
Denmark; 2Biomarker
Discovery, R&D, EXIQON A/S, Denmark; 3Molecular
Biology, EXIQON
A/S, Denmark; 4Cardiology Stem Cell Laboratory, University
Hospital
Rigshospitalet, Denmark; agap@life.ku.dk
MicroRNAs are
small, non-coding RNAs
that are known to regulate the gene expression at post transcriptional
level.
MicroRNAs are believed to play an important role in the control of
various
developmental and physiological processes. Central nervous system
(CNS),
developing brain in particular, is proved to host an impressive
diversity of
microRNAs. Most of the microRNA expression profiling is done in humans
or mice
however there is a need for information within microRNA biology in
other
mammals. Domestic pigs are considered as good animal models for human
related
neurological studies because pigs brain development and brain growth
curve is
very similar to humans. Moreover, relatively small number of microRNAs
is
annotated in domestic pig, and most of them are in silico predicted. In the
present study, microRNA
expression profiling with use of microRNA microarray and qPCR was
performed on
porcine developing brain. Our results show that microRNA expression is
regulated in tissue as well as in developmental stage specific manner.
We have
found numerous tissue and developmental stage specific microRNAs in the
pig brain.
The different levels of expression of particular microRNAs in foetal
versus
postnatal samples, confirms their important role in the regulation of
developmental and physiological processes that take place along the
brain
development. Furthermore this study supports the belief that microRNAs
act as
posttranscriptional switches that regulate gene expression when
required. P061 Highly
sensitive and specific LNA™-enhanced real-time PCR system for microRNA
expression analysis Exiqon A/S, MicroRNAs
(miRNAs) comprise a family
of highly conserved small non-coding RNAs (~22 nt). As regulators of
post-transcriptional gene expression, miRNAs play an essential role in
many
parts of development, differentiation, and physiological processes. The
accurate and specific expression analysis of miRNAs is complicated by
their
short length and sequence similarities between miRNAs in the same
family. The
specific quantification of a 22 nt sequences with single nucleotide
mismatches
is a significant challenge. We have developed a highly sensitive
real-time PCR
method for quantification of miRNAs. One of the advantages of the
Locked
Nucleic Acid (LNA™) technology is that very short high-affinity
miR-specific
primers can be designed, thus working under general PCR conditions. The
LNA™
primer design enables a simple and robust two-step method employing two
different miRNA-specific primers: a miRNA-specific RT primer is
employed in the
first-strand cDNA synthesis, and for the following SYBR®
Green-based
quantitative PCR detection, a LNA™-enhanced primer targets the miRNA
sequence
at the opposite end. Hence, the method offers accurate quantification
of
specific miRNAs directly from total RNA. The miRCURY LNA™ microRNA PCR
system
offers a high dynamic range with a linear readout of miRNA
concentrations
spanning more than 8 orders of magnitude, enabling detection of as few
as 10
RNA copies. The LNA™ enhanced primers allow discrimination between
closely
related miRNAs and between mature and precursor miRNAs. P062 microRNA
expression analysis by LNA™-enhanced real-time PCR Exiqon A/S, MicroRNAs
(miRNAs) comprise a family
of highly conserved small non-coding RNAs (~22 nt). As regulators of
post-transcriptional gene expression, miRNAs play an essential role in
many
parts of development, differentiation, and physiological processes. It
is now
established that altered miRNA expression profiles are associated with
a number
of different diseases including heart disease, neurological disorders
and human
cancers. This suggests the use of miRNAs as a novel class of biological
important biomarkers for disease diagnosis and prognosis. The study of
expression and
functional effects of miRNAs is complicated by their small size and
limited availability
of sample. We have developed a highly sensitive real-time PCR method
for
quantification of miRNAs. The LNA™ primer design enables a simple and
robust
two-step method employing two different miRNA-specific primers: a
miR-specific
RT primer is employed in the first-strand cDNA synthesis, and for the
following
SYBR® Green-based quantitative PCR detection, a LNA™-enhanced
primer targets
the miR sequence at the opposite end. The miRCURY LNA™ microRNA PCR
system
offers the possibility for highly sensitive and specific quantification
of
miRNA expression levels from total RNA. In addition,
a panel of endogenous control
primer sets has been developed for accurate and sensitive
quantification of
small nucleolar RNAs, U6 snRNA and 5S rRNA. These can be used as
normalizers,
allowing comparison of miRNA expression levels over a range of
different
samples. However, the endogenous controls must always be empirically
validated
for each study. Even very small changes in miRNA expression levels,
e.g. in
comparing different disease stages, might be biologically significant.
Reliable
normalization is therefore critical when analyzing differences in miRNA
expression. We show an example of how the use of a poor normalizer can
lead to
incorrect conclusion when studying differential miRNA expression. Finally, we
demonstrate the
excellent correlation between miRNA expression analysis using miRCURY
LNA™
Arrays and the miRCURY LNA™ microRNA PCR system. P063 Influence
of total RNA integrity on microRNA quantitation TUM,
Physiology The analysis
of RNA quality is a
valuable tool in classical gene expression profiling via qRT-PCR and
microarray
analysis. This technique may also be integrated in the routine analysis
of new
applications like the investigation of miRNA expression. Agilent
Technologies
offers a new application for the 2100 Bioanalyzer making it possible to
analyze
small RNA with the lab-on-a-chip technology. By now this chip provides
the only
possibility to quantify miRNA in absolute amounts [pg] and as a
percentage of
small RNA [%). Ongoing RNA degradation is accompanied by the formation
of small
RNA fragments and though possibly influences the miRNA quantitation
leading to
an overestimated miRNA amount. Aim of the study was to investigate the
influence of artificially caused RNA degradation on miRNA quantitation
and
miRNA expression. RNA and miRNA
were extracted from
different bovine tissues [liver, muscle, white blood cells (WBC)] in
six replicates
(n=6). Total RNA quantification was done using the NanoDrop. RNA
quality
analysis and miRNA quantitation were done with the 2100 Bioanalyzer.
The
measurement of the gene expression pattern for mRNA and miRNA was
undertaken
with real-time qRT-PCR. Results for
miRNA quantitation
showed a highly significant increase in apparent miRNA percentage
following
ongoing RNA degradation for all investigated samples. A significant
rise in
apparent miRNA concentration was demonstrated for liver and WBC
samples. Gene
expression analysis showed a highly significant negative correlation
for the CT
values and the RIN. In
conclusion, the determination of
miRNA quantity with the Bioanalyzer is just reliable for samples with
good RNA
quality. With increasing total RNA degradation level an overestimation
of the
miRNA amount occurs. The performance of miRNA qRT-PCR is dependent on
the
template quality as well as the performance of mRNA qRT-PCR. Diagnostic &
Molecular Markers (agri-vet)
Detection
and quantification of bovine, ovine and caprine milk percentages in
dairy and
soybean products using isoelectric focusing of gamma-caseins and
Real-time PCR BOKU -
Universität für Dairy
products made from ewes’ and
goats’ milk are of considerable economic importance. However, the
substitution
of these milks for cows’ milk is a fraudulent practice in the dairy
industry.
The seasonal changes and the much lower milk yield of ewes and goats,
together
with the much lower price of bovine milk are the main reasons for this
adulteration. Consequenctly, an adequate methodology is required to
control authenticity
of dairy products. Moreover, soybean dairy-like products, which are an
alternative for people suffering from an allergy against milk proteins,
have to
be checked to prevent the potential adulterations resulting from the
addition
of casein and/or whey proteins to these products and their adverse
effects on
allergic people. The
objectives of this study were
the qualitative detection as well as the quantitative determination of
cow’s
milk percentage in dairy and soybean products. Standard mixtures of
milk from
different species as well as model cheeses of different ages were used
as
references. Species identification was performed using different
electrophoretic methods, and by conventional polymerase chain reaction
(PCR) as
well as quantitative real-time PCR using species-specific primers.
Applied
methods were evaluated regarding their applicability for the detection
and
quantification of cows’ milk in mixed cheeses. In addition, soybean
milk was
spiked with different amounts of bovine milk to enable quantitative
analysis of
cow’s milk percentages in soybean products. Urea-polyacrylamide
gel
electrophoresis of caseins was restricted to the adulteration control
of milk
only. The official EU reference method (No 213/2001), which is based on
the IEF
of gamma-casein fractions, was a reliable tool to detect cows’ milk
even in
matured cheeses made from milk of other species. Usually, the this
method is
performed as a qualitative technique using reference samples, which are
certified with 0% and 1% of cow’s milk, respectively. However, after
densitometric evaluation of gamma-caseins, a quantitative estimation of
cow’s
milk percentage was obtained in mixed cheeses. Conventional PCR was
shown to be
a qualitative method, although a certain semi-quantitative estimation
could be
achieved in some cases. Real-time PCR proved to be a high-sophisticated
technique, which enables the quantitative determination of the cow’s
milk
percentage in mixed cheeses manufactured from milk of different
species, but
turned out to have an unexpected high error probability. This was
probably due
to the fact that DNA-based methods are to be applied for quantitative
adulteration control of mixed cheeses with extreme care, only! Thus,
analytical procedures used
were appropriate for the qualitative detection of cows’ milk in dairy
and
soybean products. However, quantitative results in adulteration control
have to
be understood as approximate values, and authentication of mixed
cheeses still
remains a challenge for food analysts. P065 Development
and transferability of medic EST derived SSR markers across four food
grain
legume species. 1Department
of Biotechnology and Plant Breeding, College of Agriculture, Ferdowsi
University of Mashhad, P.O.Box 91775, Mashhad, Iran; 2Pulse
Germplasm Enhancement Research, South Australian Research and
Development
Institute, GPO Box 397 Adelaide, South Australia, 5001; 3School
of
Agriculture, Food and Wine, Waite Campus, The University of Adelaide,
Glen
Osmond, South Australia, 5064; abagheri@um.ac.ir
Expressed
sequence tags (ESTs)
derived SSRs or EST-SSRs are valuable markers which can be generated
and used
from closely related genera. For plants such as grain legumes species
lacking
extensive genomic DNA sequence data, using these markers would be an
economical
method for the development of markers for diversity analysis. This
study
reports the development of SSRs from large EST databases of Medicago
truncatula
and evaluates the possible transferability across 10 genotypes of each
of four
food grain legume species namely Cicer arietinum; Lens culinaris; Vicia
faba
and Pisum sativum. Ests in the medicago database were searched from
NCBI´s
dbEST and 30 PCR primers were designed based on di-, tri and tetra
simple
sequence repeats (SSR) motifs and tested on 10 genotypes of each
species
together with four genotypes of medics. Polymorphisms were evident both
at the
genotype level and also between legume species. Thus, the study
provides
insights about those EST-derived SSRs in grain legume species that are
useful
for marker development due to their polymorphism and transferability. P066 qPCR
monitoring of cholesterol synthesis regulation Lehrstuhl
für Physiologie, TUM, Introduction:
Milk cholesterol level
regulation is a complex mechanism involving the intervention of many
metabolic
pathways, including absorption in the intestine, liver and mammary
gland
biosynthesis and transport mechanisms through blood and cellular
membranes. In
vegetarian organisms it can be hypothesized that, as diet contains
scarce
amounts of this component, the de-novo synthesis pathways play the
crucial
regulation step in the whole homeostasis.This study aims at the
characterization of the liver cholesterol synthesis and its regulation
during
the lactation cycle of Bos taurus by using qPCR. Materials and
methods:Liver biopsies
were obtained from 16 adult Brown Swiss cows at different time points
before
and after parturition (weeks -2, 0, 2, 4, 8) and RNA extracted. After
reverse
transcription, the expression of three rate limiting genes from the
cholesterol
synthesis route (HMGCoA-reductase, HMGCoA-synthase and FDFT) and three
regulation factors (SREBP1, SREBP2 and SCAP) was measured by qPCR. All
experiments were performed using the LightCycler SYBRGreen technologie
(Roche
Diagnostic). Expression data was normalized using three housekeeping
genes in
form of a normalization index (Ubiquitin, GAPDH and ß-actin). Results: A
coordinated regulation of
gene expression at the onset of lactation was demonstrated, most
probably as an
image of the immense changes in metabolism of this crucial molecule
that the
bovine organism has to face immediately after parturition. A
significant
up-regulation of the SREBP resgulation pathway occured at the first
stages
after parturition (weeks 0 and 2) and was followed by the correspondent
increase in synthesis enzymes HMGCoA Reductase and FDFT expression
during the
following time points (weeks 2 to 8). HMGCoA synthase showed similar
tendencies, although non-significant and non-correlated with the other
studied
factors, so it can be hypothesized that this crucial enzyme is
subjected to
different regulation mechanisms. P067 Development
of a real-time PCR for detection of Mycoplasma felis in cats National
Veterinary Institute (SVA), Infection
with Mycoplasma felis is
associated with conjunctivitis, respiratory tract disease and arthritis
in
domestic cats. To enable intervention with antimicrobials targeting
mycoplasma,
accurate and rapid diagnosis of these infections is necessary. To this
end we
have developed a real-time PCR protocol for detection of Mycoplasma
felis based
on dual-labeled fluorogenic probe technology targeting the gene for
elongation
factor Tu ( tuf ). Sequencing this gene in the type strain
(CO) and
clinical samples of M. felis as well as type strains of other
mycoplasma
species allowed us to target regions that were species-specific while
highly
conserved within M. felis . Using the same panel of mycoplasma
type
strains and clinical strains of feline origin to evaluate the developed
assay,
we found a detection limit in the order of 10fg of M. felis genomic
DNA
and no cross-reaction with other mycoplasmas. We also compared the
assay to an
available conventional PCR on a panel of conjunctival swabs from cats
with
clinical signs of ocular infection (n=100). We found the new assay to
be more
sensitive and specific in this setting, detecting one additional
positive while
rejecting a sample found positive by conventional PCR which carried a tuf
gene
identical to that of the type strain of Mycoplasma cynos (H831).
We
suggest that the developed assay might be a useful tool for research
and
routine diagnostics. P068 Differential
temporal expression of staphylococcal enterotoxins and enterotoxin-like
superantigens during in vitro growth AFSSA (French
Food Safety Agency),
France; s.derzelle@afssa.fr Staphylococcal
food poisoning (SFP)
is an intoxication resulting from ingestion of foods containing one or
more
preformed enterotoxins from Staphylococcus aureus. Staphylococcal
enterotoxins
(SEs) and enterotoxin-like proteins (SEls) are a family of structurally
related
proteins belonging to the pyrogenic toxin superantigen family. To date,
21types
have been described (SEA-SEE, SEG-SEI, SElJ-SElV). To better
understand how those genes
are expressed during a growth cycle, we developed a qRT-PCR procedure
to
determine their temporal expression pattern. PCR assays that allow for
the
screening of 18 se/sel genes were designed and a panel of strains was
examined
for their distribution. A total of 28 strains displaying various
combinations
of se/sel genes were selected for further kinetic mRNA study. To examine
the impact of growth
phase on the dynamics of se/sel transcription, strains were cultured at
37°C in
BHI broth and RNA harvested at 4 time points from mid-exponential to
late
stationary phase. Expression of each gene was measured by qRT-PCR and
relative
expression ratios were calculated. Three reference genes whose
expression was
found to be few affected by growth conditions were selected with the
GeNorm
software and quantified at the same time. Normalization was provided
using the
geometric mean of these latter genes. Different
patterns of expression
were found depending on the species and genes studied. seb and sec mRNA
abundance were found to be transiently upregulated at the transition
into
stationary phase, a characteristic of their regulation by the accessory
gene
regulator (agr) system. sed was also significantly induced at the end
of the
exponential growth phase but in lesser extend. In contrast, most se/sel
transcript levels remained relatively constant or decreased from
exponential to
stationary growth phases. P069 Expression
of VEGF-B isoforms and neuropilin-1 in bovine ovary during different
physiological stages Physiologie
Weihenstephan,
Technische Vascular
endothelial growth factor-B
(VEGF-B) is a member of the VEGF family, a system of growth factors
that is
known to regulate blood vessel angiogenesis. Neuropilins (nrp.-1 and
nrp.-2)
are co- receptors for VEGF-R and can thus enhance VEGF effects. VEGF-B
can only
bind to nrp.-1. The aim of this study was to characterise the
expression
patterns of the mainly anti-apoptotic factor VEGF-B and its splicing
isoforms
(VEGF-B167, VEGF-B186) in bovine ovary during final follicle
maturation, corpus
luteum (CL) function and regression (induced luteolysis) as well as the
expression of nrp.-1. Experiment 1: Antral follicle classification
occurred by
follicle size and oestradiol-17β (E2) concentration in follicular fluid
(FF)
into 5 groups (<0.5, 0.5-5, 5-40, 40-180 and >180 ng/ml).
Granulosa cells
(GC) and theca interna (TI) were investigated separately. Experiment 2:
CL were
assigned to the following stages; days 1-2, 3-4, 5-7, 8-12, 13-16,
>18
(after regression) of estrous cycle and of pregnancy (month 1-2, 3-5,
6-7,
>8). Experiment 3: Induced luteolysis, cows on days 8-12 were
injected with
PGF2α analogue and CL were collected by transvaginal ovariectomy before
and
0.5, 2, 4, 12, 24, 48 und 64h after PGF2α injection. The expression of
mRNA was
measured by qRT-PCR and the concentrations of the hormones in FF by
ELISA. In
GC and TI of antral follicles only VEGF-B186 could be measured. Its
mRNA levels
in GC increased significantly in larger follicles. The same expression
patterns
could be observed for nrp.-1. In the TI VEGF-B186 was constantly up
regulated
during follicle maturation, but declined in follicles with E2 >180
ng/ml. In
CL during estrous cycle transcripts of both VEGF-B isoforms declined
from days
3-4 to 13-16, but were up regulated significantly in CL >18 days.
During
pregnancy mRNA of both isoforms were constantly expressed. During
induced
luteolysis VEGF-B186 increased significantly 24h after PGF2α injection,
while
nrp.-1 decreased at this point of time. Both factors showed constantly
high
expression during functional luteolysis. In conclusion, the temporal
mRNA
expression of VEGF-B isoforms and nrp.-1 in antral follicles and CL of
induced
luteolysis, as well as VEGF-B isoforms in CL of estrous cycle and
pregnancy,
suggests them to be important mediators of follicle maturation as well
as CL
formation, function and regression in bovine ovary. P070 Direct
Quantification of thermophilic Campylobacter species in chicken rinse
samples
using real-time PCR Bavarian
Health and Food Safety
Authority, Quantitative
microbial risk assessment
is becoming more and more important in diagnostics of food-borne
pathogens. It
can be based on the existence of virulence factors or simply on the
number of
pathogens per food. It has been shown that conventional microbiological
methods
are not suitable for quantification because of low sensitivity,
influences of
background flora and labor-intensity. Especially if bacteria are
difficult to
culture counting of colony-forming units is not reliable. Using
real-time PCR
for quantification, however, the number of cells can be determined more
reliably as each cell, dead or alive, will be detected. One of the most
common
causes of food associated bacterial gastroenteritis worldwide are
thermophilic
Campylobacter species. Therefore, quantitative risk assessment of these
bacterial species is of great importance. In this study, 44 chicken
rinse
samples were analyzed. Presence and number of cells of Campylobacter
spp. were
determined without pre-enrichment of the samples applying plate
counting in
comparison to real-time PCR. A published real-time PCR assay that
specifically
detects C. jejuni, C. coli and C. lari was applied. For quantification
of cell
numbers inactivated and stabilized Campylobacter cells were used as
reference
material for the quantitative PCR. This reference material is used for
the
calibration of the data with respect to the total efficiency for the
entire
process from DNA preparation to the quantitative real time PCR. It was
shown
that the real-time PCR was useful for quantitative detection of
thermophilic
Campylobacter spp. in chicken rinse samples. Compared to the cultural
method,
the results are provided faster and can be performed independently of
the
background flora present in the sample. The application of a simple to
use
precalibrated standard with known cell number accurately determines the
number
of pathogens in a food sample. P071 Effect
of apple pectin on gut microbiota - qPCR in applied microbiology 1Technical This study
was part of the large
European project ISAFRUIT aiming to reveal the biological explanations
for the
epidemiologically well-established health effects of fruits. The
objective was
to identify effects of apple and apple product consumption on the
composition
of the cecal microbial community in rats, as well as on a number of
cecal
parameters, which could be influenced by a changed microbiota. Principal
Component Analysis (PCA)
of cecal microbiota profiles obtained by PCR-DGGE targeting bacterial
16S rRNA
genes showed an effect of whole apples in a long-term feeding study (14
weeks),
while no effects of apple juice, purée or pomace on microbial
composition in
cecum were observed. Administration of pectin derived from apples
resulted in
considerable changes of these DGGE profiles. A 2-fold
increase in the activity of
beta-glucuronidase was observed in animals fed with pectin (7% in the
diet) for
four weeks, as compared to control animals (P<0.01). Additionally,
the level
of butyrate measured in pectin-fed animal was more than double of the
corresponding level in control animals (P<0.01). Sequencing revealed
that
DGGE bands, which were suppressed in pectin-fed rats, represented
Gram-negative
anaerobic rods belonging to the phylum Bacteroidetes, whereas bands
that became
more prominent represented Gram-positive anaerobic rods belonging to
the phylum
Firmicutes, and specific species belonging to the Clostridium Cluster
XIVa.
These findings indicate that consumption of apple pectin increases the
population of butyrate- and beta-glucuronidase producing Clostridiaceae
in the
rat gut. The
quantitative changes in bacterial
composition were verified by quantitative real-time PCR using
SybrGreen. All
results were calculated as ratios of relative expression levels to HDA
expression levels in order to correct data for differences in total DNA
concentration between individual samples.The HDA primer set amplifies
the V2-V3
region of the 16S ribosomal DNA gene, a well conserved bacterial marker
region. The RT-PCR
data will be presented at
the symposium as an example of the application of quantitative PCR in
the data
analysis of prokaryotes. P072 Expression
of estrogen receptor alpha and beta in reproductive tissues of male
growing
piglets TU Objectives:
Exposure to estrogens is
critical during juvenile development in males when low endogenous
hormone
levels prevail. The sensitivity towards sex steroids might be of
specific
relevance for disturbances associated with subsequent reproductive
failure. We
characterized the expression of estrogen receptors alpha (ESR1) and
beta (ESR2)
during different points of time in male pre-pubertal piglets. Methods: Male
siblings of German
Landrace sows (n=6) were slaughtered at <1h, 11 and 48 days after
birth.
Estradiol-17β (E2) concentration in plasma at slaughtering was measured
using a
competitive ELISA. Testis, corpus epididymis and prostate tissue were
sampled.
RNA was extracted for quantitative real-time PCR of ESR1 and ESR2. ESR1
protein
was determined by immunohistochemistry. Results: E2
concentration was
highest in newborns (78.3±14.8 pg/ml), intermediate at the age
of 11 days
(9.2±2.2 pg/ml) and lowest in pre-pubertal piglets
(1.8±0.5 pg/ml). ESR1
transcripts were detected in all tissues analyzed. ESR1 transcript
abundance in
corpus epididymis was 2.1-fold and 1.7-fold on days 11 and 48, compared
to
newborns (P=0.01), while prostate and testis showed no significant
differences
over time. ESR1-protein increased in prostate from <1h to 48 days
after
birth in smooth muscle cells and glandular epithelial cells. In testis,
ESR1
was mainly localized to premature seminiferous tubules, in epididymis
to
epithelial cells. A methylation analysis of the ESR1 promoter using
Pyrosequencing-technology is undertaken attempting to explain
differential gene
expression. ESR2 transcripts were present in testis at the three
distinct time
points, but below the detection limit in prostate and epididymis at 11
and 48
days after birth, respectively. Conclusion:
In summary, growing
piglets exhibit very distinct endogenous levels of E2 over the first
few weeks
of lifespan offering an interesting model to study effects of
endogenous
estrogens on development. Male reproductive tissues are distinctly
regulated
during pre-pubertal growth with respect to ESR1, which may be
associated with
tissue-specific differences of sensitivity to estrogens. P073 1Department
of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine,
Ghent
University, Heidestraat 19, 9820 Merelbeke, Belgium; 2Centre
for
Medical Genetics Ghent, Ghent University Hospital, De Pintelaan 185,
9000
Ghent, Belgium; Mario.VanPoucke@UGent.be
Peroxisome
proliferator-activated
receptor γ coactivator 1α (PPARGC1A) is a versatile coactivator and is
of vital
importance to energy and fat metabolism. Based on these functions, it
not only
is an excellent candidate gene for meat quality in pigs, but also is of
great
interest for human research. The information on the in vivo gene
expression and
relationship between PPARGC1A and its downstream target genes is
however very
limited, especially in the pig. In this study, the gene expression
pattern of
PPARGC1A and 10 of its putative target genes was determined in backfat
and
longissimus dorsi muscle (MLD) samples from 20 pigs, by real-time PCR.
Samples
were preserved in RNA-later (Sigma-Aldrich), crushed to powder with
liquid
nitrogen and stored at -80°C until use. The Aurum Fatty and Fibrous
Tissue RNA
Isolation Kit (Bio-Rad) and iScript cDNA Synthesis Kit (Bio-Rad) were
used for
total RNA isolation and cDNA synthesis, respectively. Real-time PCR was
conducted with the Platinum SYBR Green qPCR Supermix UDG (Invitrogen)
on an
iCycler iQ Real-Time PCR Detection System (Bio-Rad). Evaluation of
reference
gene expression stability by geNorm indicated that normalisation with
ACTB, TBP
and TOP2B provided reliable mRNA expression results. Except for
UCP3 and LPL, a very
significant expression difference was found between backfat and MLD for
all
genes (P < 0.01). Statistical analysis indicated that there was a
strong
genetic regulation of the mRNA expression of several target genes. A
positive
correlation with PPARGC1A gene expression was found for CPT1B, GLUT4,
PDK4 and
TFAM (P < 0.0001). A negative correlation was found for UCP2, FABP4,
LEP (P
< 0.0001), and TNFα (P = 0.0071). No significant correlation was
detected
for UCP3 and LPL. In conclusion, these data suggest a clear impact of
PPARGC1A
on energy and lipid metabolism in vivo in the pig through several of
these
downstream target genes. P074 High
Resolution Melt analysis of _Brachyspira_ for identification of B. hyodysenteriae and B. pilosicoli Technical We will show
a new High Resolution
Melt (HRM) assay for the identification of the genus Brachyspira by
preamplification with primers specific for B. hyodysenteriae, B.
pilosicoli,
B. murdochii, B. Innocens, B. intermedia and “B. suanatina” and
subsequent HRM analysis to differentiate between B. hyodysenteriae,
B.
pilosicoli and all others. The methodology is simple and straight
l PBS andmforward,
one loop full of bacteria is suspended in 200 boiled
for ten minutes, the lysate is centrifuged at 10
000 x g for 10
minutes and the supernatant is transferred to a clean tube. To
investigate the
potential of the assay in routine diagnosis of pig diarrhoea, stool
samples
were spiked with different concentrations of Brachyspira cells.
Total
DNA from the stool samples were extracted with QIAamp DNA Stool Mini
Kit with
the QIAcube (QIAgen). DNA from reference strains were extracted with
Easy-DNA
(Invitrogen). All DNA samples were analysed with the same pair of
primers
giving an 88 bp amplicon from 23S rDNA in RotorGene 6000 (Corbett).
Different
dilutions of reference strains were used as positive controls of the
different
species in each run. The melt and
HRM analyses of samples
were visually compared to the melting plots and HRM analyses of
reference
strains. Brachyspira DNA will result in three melting peeks
which can be
identified as belonging to B. hyodysenteriae, B. pilosicoli or
other Brachyspira.
sp. The normalised plot of the HRM analyses shows three separate
curves.
Reaction efficiencies and detection ranges for individual Brachyspira
species
as well as the validation regarding negative controls are shown. The
Danish
Veterinary Institute is presently engaged in developing new methods for
the
diagnosis of the causative agents in pig diarrhoea. The HRM assay has
the
promise of becoming a valuable diagnostic tool in the laboratory. P075 LUX
PCR assay and comparison with SYBR-GREEN PCR and TaqMan PCR formats for
detection of DNA virus (PCV2) At present
several formats of
real-time PCR were developed with application for diagnostics assays of
infectious diseases in human and veterinary medicine. The LUX PCR
(light upon
extension PCR) is relatively new technique of real-time PCR which is
based on
the use of two primers where only one primer is labeled near 3’-end
with a
fluorophore molecule. We have developed for the first time LUX PCR
assay for
the detection of porcine circovirus type 2 (PCV2) infecting pigs. LUX
PCR assay
amplifying a 119 bp fragment of PCV2 ORF1 detected at least 20 copies
of viral
DNA inserted into recombinant plasmid. The dynamic range of this
quantitative
assay covered range from 200 to 100 000 000 copies of viral genome. The
assay
specifically detected only PCV2 with negative results for other swine
viruses
(CSFV, PRRSV, PCV1). The
SYBR-GREEN PCR with primers
CF8/CR8 (1) amplifying a 263 bp fragment and TaqMan PCR system (2)
modified in
our laboratory amplifying a 64 bp fragment were used for the comparison
with
LUX PCR assay. The assays were tested on clinical samples from PCV2
infected
pigs. DNA was isolated from samples by the Chelex method. The
amplification
curves were characteristic for different types of real-time PCR
techniques and
Ct values for the same sample were also different from assay to assay.
The
lowest numerical Ct values were obtained in SYBR-GREEN PCR, than in
TaqMan PCR,
the highest Ct values were observed for LUX PCR. This observation
corresponds to
basic principle of the detection of fluorogenic signal during
amplification
process. However the threshold values in all three assays provided
linear
calibration curve with similar quantification of viral copies for a
particular
sample (tested in range 100 to 10 000 copies/25 ul of assay volume).
Reproducibility of results tested in inter-experiment evaluation was
relatively
stable varying not more than a half order of viral copies. Negative
samples
provided values not more than 0 to 40 copies (in SYBR-GREEN and TaqMan
PCR).
All these data suggest that the evaluation of results for a particular
real-time PCR assay should be carefully interpreted. LUX PCR and TaqMan
PCR
systems are in principle, and in our experiments too, more specific
than
SYBR-Green PCR. LUX PCR as methodologically simple technique is a
method of
choice for the laboratory detection and quantification of PCV2 in
clinical
samples. References: 1.Larochelle,
R. et al.: Journal of
Virological Methods 80, 69-75, 1999. 2.Blanchard,
P. et al.: Journees de
la Recherche Porcine en This work was
supported by 6.
Framework Program EU No. NMSACC-PCVD 518432, APVV-20-019605 and VEGA
No.
1/0389/08 Optimisation
of a qRT-PCR assay to determine Vp1 expression in relation to
pre-harvest
sprouting tolerance in triticale 1Faculty
of Biosciences and Landscape Architecture, University College Ghent and
Ghent
University, Belgium; 2Institute for Agricultural and
Fisheries
Research - Plant Unit, Belgium; 3Faculty of Bioscience
Engineering,
Ghent University, Belgium; sarah.delaethauwer@hogent.be
Pre-harvest
sprouting (PHS) is a
major problem in triticale production during rainy harvest periods,
inducing a
severe loss in grain yield and quality. Due to the influence of
genotype, the
environmental conditions and their interaction, the only solution to
this
problem is to breed PHS tolerant cultivars. Several field-independent
methods
exist but seem to be unreliable to select for PHS tolerant triticale
varieties.
The aim of this study is to obtain more insight in the complexity of
PHS
through a molecular genetic approach. Typically PHS
sensitive varieties
lack adequate dormancy levels to prevent early sprouting during wet
harvest
periods. In literature many genes are cited for their important role in
embryo
development, dormancy induction and dormancy maintenance. These genes
are
mostly involved in pathways of plant hormone synthesis and catabolism
or
alpha-amylase synthesis and degradation. Most of these genes are
genetically
analysed in wheat, rice, barley, maize, rye or Arabidopsis thaliana
and
may be suitable for use in a qRT-PCR assay. As dormancy is installed
during
kernel development, an integrated study during kernel development may
reveal
some factors which can be useful to select for PHS tolerant varieties. Plants of a
PHS sensitive and a PHS
tolerant variety of both wheat and triticale were grown in a growth
chamber
under controlled conditions from flowering to ripeness. At regular days
post
anthesis kernels were harvested for germination tests, plant hormone
detection
and RNA extraction. After RNA extraction and DNase treatment first
strand cDNA
was prepared and could be used for qRT-PCR with SYBR Green chemistry.
For a
relative quantification of the expression level of the intended target
genes, a
proper selection of reference genes is necessary for an adequate
normalisation.
In this study eleven reference genes were selected and checked for a
stable
expression pattern during kernel development in both wheat and
triticale. The
four reference genes with most stable expression pattern were selected
using
the GeNorm application. The first results indicate that the genes
coding for
beta-tubulin, ubiquitin conjugating enzyme, elongation factor and
cytoplasmic
malate dehydrogenase perform the most stable expression level and can
be used
as references. As a first
gene of interest many
attention was paid to the ‘viviparous’ Vp1 -orthologue. In
wild oat and
wheat a correlation between the expression level of the Vp1 gene
and the
level of seed dormancy is described. However, until now our study could
not
confirm this correlation in mature seeds with use of the qRT-PCR
technique,
probably due to a low expression level of Vp1 in mature
embryos. The
above described alternative approach may contribute to a better
understanding
of the expression profile of Vp1 and other target genes during
kernel
development of triticale specifically, and PHS in general. P077 Expression
of some angiogenic factors in bovine follicles before and after LH surge Physiology
Weihenstephan, TUM, Angiogenesis
is defined as the
generation of new blood vessels through sprouting from already existing
blood
vessels in a process involving different angiogenic factor families.
The aim of
this study was to evaluate the expression patterns of hypoxia-inducible
factor-1 alpha (HIF), VEGF and angiopoietin (ANPT) system members (VEGF
isoform
120, 164, 188, VEGFR-1, VEGFR-2, ANPT-1, ANPT-2, and Tie-1, Tie-2
receptors) in
time defined follicle classes before and after GnRH application and
after
ovulation in cow. Ovaries containing preovulatory follicles or new
corpora
lutea (CL) were collected at 0h, 4h, 10h, 20h, 25h (follicles) and 60h
(CL day
1-2) relative to injection of GnRH (n=5/group). For better
characterization of
follicle classes, estradiol-17beta and progesterone were determined by
EIA in
follicular fluid (FF). The mRNA of HIF decreased in follicles only
during the
periovulation phase, followed by a significant increase in early CL
tissue.
Transcripts of all VEGF isoforms were upregulated 4h after GnRH
followed by
lowest expression around ovulation. The VEGF peptide content in FF
increased
25h after GnRH. All VEGF isoforms as well as their receptors were
upregulated
again after ovulation. ANPT-1 mRNA decreased significantly in follicles
during
LH surge. ANPT-2 decreased 10h after GnRH and in the follicle group
during
ovulation. Tie-1 and Tie-2 mRNA expression decreased in follicle group
during
ovulation, with a further increase in early CL tissue. It is likely
that the
decrease of ANPT-1 and therefore the increase of the ANPT-2/ANPT-1
ratio during
the LH surge is a basic mechanism of vascular remodelling in follicles
during
periovulation. In conclusion, the temporal expression pattern of
angiogenic
factors (HIF, ANPT and VEGF family members) during periovulation
suggests them
to be important mediators of the ovulatory process and the early CL
formation
(angiogenesis) in the bovine ovary. P078 1TIMAC
AGRO Introduction –
Verticillium wilt remains one of
the most serious soil borne diseases worldwilde. An array of different
strategies to reduce the consequences of pathogen pressure is
available. Among
these methods, application of natural compounds, which stimulate
locally and
systemically plant defense reactions, is becoming an industrial
alternative to
the application of chemicals with deleterious side effects. Our
objective was
to investigate, through the quantitative and very sensitive real-time
RT-PCR
technology, the potential of Coactyl™, composed of humic acid
associated to a
phenolic acid, to act as a plant defense inducer conferring resistance
to
Verticillium dahliae wilt in tobacco used as a model. Preliminary
experiments
showed that application of Coactyl drastically reduced the severity of
V.
dahliae infection, which was further quantified by real time RT-PCR. PR
protein
genes are well recognized tobacco molecular markers of defense
activation.
There are more than 10 families of these proteins, which exhibit
anti-fungal
and anti-microbial activities. Expression of a representative set of
these
genes, PR1 acid, PR2 acid and basic isoforms, PR3 basic and PR5 acid
and basic
isoforms, was evaluated by real-time RT-PCR. Results - Quantification
by real-time PCR of
_V. dahliae _growth in planta was achived using specific primers of the
internal transcribed spacer ITS of V. dahliae (AB353346).
Sequencing
amplicon clones identified only the target V. dahliae sequence and no
tobacco
sequence demonstrating the specificity of the reaction. Experimentally,
tobacco
plants were treated by Coactyl 10 days before _V. dahliae _inoculation.
Then,
seven days after inoculation, V. dahlia was quantified in roots, stems
and
leaves of infected and mock-treated plants. We measured a severe
reduction of
_V. Dahliae _amounts as well as a delay is plant colonization. As
expected from
the sensitivity of the method, _V. Dahliae _could be detected before
any
symptom could be observed. Decrease in _V. dahlia _infection correlated
with
the increased expression levels of PR protein genes, locally in roots
were
Coactyl was applied, and systemically, in stems and leaves. Conclusion - First, the
use of the real-time
(RT-PCR/ PCR) technologies allowed to asses both the level of fungus,
thus
quantifying the level of disease resistance, and the level of plant
defense
genes, thus quantifying the level of plant defense mechanisms. Second,
our
results highlight the potential of Coactyl as a novel compound able to
provide
commercially significative resistance of tobacco against _V. dahliae
_trough
the activation of molecular defense responses. Preliminary disease
resistance
tests have shown that Coactyl is also active in other dicotylodenous
plants
further supporting Coactyl as a novel bioactive preparation. The
molecular mode
of action of Coactyl remains to be determined, however. It is currently
under
investigation. P079 Rapid
detection and differentiation of foodborne pathogens in the official
food
control using real-time-PCR Bavarian
Health and Food Safety
Authority, The rapid
detection and
differentiation of human pathogens in food is one of the main
instruments of
the official preventive consumer protection policy. The cultural
detection of
foodborne pathogens is a time-consuming and ineffective method for
testing
large numbers of food or environmental samples, especially in outbreak
situations and microbiological methods are not able to deliver
informations
about different toxin gene combinations of humanpathogenic bacteria
like
Clostridium botulinum, Clostridium perfringens, Bacillus cereus or
Vibrio spp.
But these informations are essential for a well-founded risk assement,
especially for bacteria species, which exist in a pathogenic and a
non-pathogenic variant. These are the
reasons, why in the
last few years the Bavarian Health and Food Safety Authority has
established
multiplex-real-time-PCR systems not only for the detection of the
“classical”
foodborne pathogens like Salmonella spp. or EHEC, but also for the
rapid
detection and differentiation of Clostridium botulinum, Clostridium
perfringens, emetic and diarrhoeic Bacillus cereus and pathogenic
Vibrio spp..
The methods are used for screening without enrichment directly from the
sample
material, for screening after enrichment or for verification and
differentiation
of the isolates. Every multiplex real-time-PCR is combined with an
heterologous
Internal Amplification Control system based of the pUC 19-plasmid.
Depending on
the sample material and the kind of bacteria different DNA extraction
methods
are applied in routine diagnostic. Data collected in the diagnostic of
gram-positive bacteria (Clostridium spp. and Bacillus spp.) from
different
sample material using two commercial extraction systems will be
presented. P080 Stage specific transcription Studies on Latrophilin-like protein 2 of the cattle strongyle Cooperia oncophora using Quantitative Real-time PCR1University
of The new
anthelmintic compound
emodepside has a wide range of efficacy and also resistance breaking
properties. The G-protein coupled receptor (GPCR) HC 110-R of the sheep
nematode H. contortus has been identified previously as a
putative
target. It’s orthologue in C. elegans is latrophilin-like
protein 1
(LAT-1). Another latrophilin-like protein, LAT-2, is discussed as an
additional
receptor for emodepside in C. elegans . We identified the
orthologous
GPCRs in a subset of parasitic nematodes. Recently a dose-dependent
effect of
emodepside on worm development in C. elegans was shown. Also reduced
emodepside
sensitivity, measured via a locomotion assay, in early larval stages
(L1 – L3)
was detected. The time required for hatching of eggs exposed to
emodepside did
not significantly increase or decrease. Own studies showed via egg
hatch test,
that hatching is not affected in several gastrointestinal nematodes.
With a
view to possible differences in efficacy on different developmental
stages, we
analysed the transcription level of LAT-2 in eggs, mixed first and
second stage
larvae, infectious third stage larvae, adult males and females of the
cattle
strongyle C. oncophora . We applied 18 S rRNA, 60 S acidic
ribosomal
protein, ß-tubulin and Glyceraldehyde-3-phosphate dehydrogenase
as reference
genes for the assay. There were no significant differences to detect in
the
transcription level of LAT-2 across the five examined developmental
stages of C.
oncophora . This shows that no direct correlation between a
reduced
emodepside sensitivity in eggs and early larval stages and the presence
of
LAT-2 can be created here. Whether this is due to the fact, that LAT-2
is
actually not involved in the mode of action of emodepside, or that
other
components of the signalling cascade might regulate the effect of the
anthemintic remains to be investigated. P081 Suppression
of eosinophiles after ovulation alters the progesterone values of the
bovine
corpus luteum -Evalution of FGF and VEGF gene expression via qPCR Lehrstuhl
für Physiologie / TUM, The funktion
of eosinophils (EOs) is
unknown during the development of the bovine corpus luteum (CL). We
suppressed
the migration of eosinophils into the ovary before and during ovulation
through
the injection of cortisone (Dexamethasone (D)) to evaluate the role of
EOs in
the CL. Material and methodes: 10 cows were subdivided in two groups (n
= 5).
Oestrus synchronisation with two injections of 500 µg
Cloprostenol within 10
days. 18 hours (h) later 15 mg Dexamethasone or 15 ml saline were
given.
Ovulation was induced 24 h later with 14 µg GnRH. Second
injection of 15 mg
Dexamethasone or 15 ml saline 12 h after the GnRH injection. Blood
samples from
the jugular vein were taken every day. EO were counted till day 7 every
24 h.
The ovaries were collected at day 2 and 5 of the oestrous cycle.
Protein
concentrations of progesterone, fibroblast-growth-factor-2 (FGF-2) and
vascular
endothelial growth factor (VEGF) were measured by ELISA. The gene
expression of
FGF-1, -2, -7, VEGF-120, -164, -188 and the receptors FGFR, FLK and FLT
were
evaluated by qRT-PCR using the Rotor Gene 3000. Results: EOs counted in
blood
decreased significantly after the first D injecition till day 7 with no
decrease in the control group. The progesterone values decreased
significantly
in the D group from day 8 – 17 (oestrous cycle) compared to the control
group.
FGF-2 protein level increased significantly at day 2 in the D group
compared to
day 5 and to the control group. No significant regulation was found in
the
control group at day 2 and day 5. VEGF protein level at day 2 of the D
group
decreased significantly compared to day 5 and the control group. The
expression
of VEGF-120, -164, FLT and FGF-2 were not significantly regulated.
VEGF-188
showed a significant higher expression in the D group at day 2 than at
day 5,
which was not seen in the control group. The receptor FLK was
significantly
down regulated at day 2 in the D group compared to control group. It
was also
significantly lower expressed at day 5. The mRNA expressions of FGF-1
and FGFR
were significantly down regulated in the D group at day 5 compared to
day 2.
FGF-7significantly increased in the D group at day 5 compared to day 2
and the
control group at day 5. Conclusion: The lower progesterone level in the
D group
might be caused by an undersupply of luteal cells due to a lesser
capillary
density in the developing CL induced by the lower protein level of VEGF
and the
decrease of the FLT expression. VEGF normally acts as strong angiogenic
agent
in the developing CL, which seems to be suppressed during D treatment.
FGF-2 is
known to be an inhibitory factor of apoptosis in granulosa cells. Its
high
protein level during D treatment might indicate a counter regulation
due to a
higher risk of apoptosis induced through the lesser capillary density.
It could
be possible that cows with a high cortisone blood level during
ovulation
develop an insufficient CL leading to a lower pregnancy rate. P082 The
Development of Novel Real-Time PCR Veterinary Diagnostic Assays The
development of validated, novel,
sensitive and specific diagnostic assays for the detection of microbial
and
viral pathogens is a key issue in the Agri-food industry and the
veterinary
research/animal health care sectors. Infections caused by these
pathogens
result in major financial losses and their timely identification would
serve to
limit the spread of disease. In the Molecular Virology Laboratory we
are
developing a suite of multiplex diagnostic assays, based on
dual-labeled
hydrolysis probe technology, for the detection of selected bovine
pathogens and
other species specific markers. This project focuses on key areas of
veterinary
diagnostics; bovine respiratory infections which are a critical
causative
factor in mortality in adult cattle and calves, accounting for between
24-30%
of all cases noted in Ireland (2007) and bovine abortion which
represents a
major economic loss, while also posing the risk of human zoonotic
infection.
Recent reports have indicated the involvement of numerous organisms in
this
area and disease targets selected for study include Bovine Genital
Campylobacteriosis, Infectious Bovine Rhinotracheitis, Leptospira
hardjo,
Neospora caninum, Respiratory Syncytial Virus and Bovine Para-influenza
3 virus
(PI-3). Additionally EC regulations require the species specific
identification
of all feed products for farmed animals entering the food chain; thus,
the
development of a Meat and Bone Meal composition test is also
fundamental to
this project. Assay conception and design for the detection of several
of these
targets has been completed in the lab and final validation and
comparison to
the current gold standard of these tests is currently underway. These
assays
will be rapid, economical, amenable to high-throughput and will also
further
serve as tools to research the pathology of the infections concerned. P083 Use
of real-time PCR methods targeted on the heat shock protein genes for
the
quantitation of bovine mastitis pathogens Streptococcus agalactiae,
Streptococcus uberis and Streptococcus bovis in milk and food samples. Streptococcus
agalactiae,
Streptococcus uberis, and Streptococcus bovis are the major pathogens
which
cause mastitis in dairy herds. Since conventional methods for the
detection of
these mastitis pathogens are laborious and time-consuming, rapid and
reliable
methods are needed. In this study, we developed real-time quantitative
PCR
methods using primers designed from the heat shock protein genes for
the
quantitative detection of S. agalactiae, S. uberis, and S. bovis. Using
such
methods, all the target strains could be specifically and
quantitatively
detected in milk and meat samples. As these real-time PCR methods were
used for
the direct detection of mastitis pathogens in milk and food samples,
the
detection limit was N (N= 1–9) ×102 CFU per ml or per g of the
sample. The
endogenous microflora in these samples would not interfere with the
detection.
If a 10 h pre-enrichment step was performed, the detection limit was
N×100 CFU
per ml or per g. Thus, such real time PCR quantitative methods could be
used
for the specific and quantitative determination of bovine mastitis
bacteria. P084 Validation
of a real-time PCR for quantification of Lawsonia intracellularis in
porcine
faeces samples Introduction - Lawsonia
(L.) intracellularis is the etiologic agent of porcine
proliferative
enteropathy, which occurs in different forms in pigs. In live pigs, PCR
on
faeces samples has been reported as being a reliable and suitable
technique.
While conventional PCR assays give only qualitative results, it would
be
beneficial to evaluate the amount of L. intracellularis in
faeces,
because the association between dosage and disease has been previously
published. The aim of the present study was to develop and to validate
a real
time PCR for the detection and quantification of L. intracellularis
in
porcine faeces samples. Material and Methods - Specific
target sequences
were evaluated by comparing single genes from L. intracellularis with
published sequences in the GenBank. A further set of primers was used
to
produce a larger fragment, which was flanking the target sequence 200
bp
forward and reverse. This PCR-product was cloned and used as calibrator
for
quantification by means of ΔΔCt. The validation was performed according
to
international guidelines (CVMP/VICH/590/98; GL1 & CVMP/VICH/591/98;
GL2,
Committee for Medicinal Products for Veterinary Use). The parameters
specificity, detection limit, quantification limit, linearity, range,
robustness and precision including repeatability and intermediate
precision
were assessed by testing plasmids, several bacteria and faecal samples.
Furthermore the recovery and uncertainty were evaluated. Finally, the
influence
of storage (time and temperature) of the faecal samples on the quantity
of
specific nucleotides of L. intracellularis was examined. All
reactions
included an internal positive control, were performed in triplicates
and
repeated three times. Statistical analyses were carried out with
SAS® 9.1 (SAS
Inst., P085 Differential
expression of apoptotic factors in bovine endometrium and embryos
during the
preimplantation period 1Physiology
Weihenstephan, Technical University of Munich; Bovine
trophoblasts release IFN-tau,
a type I IFN, as pregnancy recognition signal. Since type I IFNs exert
growth
inhibitory and proapoptotic actions, the expression of type I IFN
receptor
subunits IFNAR1 and IFNAR2 was analysed in the bovine endometrium and
the
respective trophoblasts during the preimplantation period.
Additionally, key
factors of the extrinsic and intrinsic apoptosis pathways were
determined.
Uteri of estrous synchronized Simmental heifers were flushed post
mortem at day
12, 15, and 18 of cycle or pregnancy for the recovery of embryos and
the
sampling of ipsilateral endometrial tissue for quantitative RT-PCR,
luminescence caspase activity assays and histochemistry. The mRNA
levels of
both type I IFN receptor subunits showed neither significant variations
in
cyclic nor pregnant animals whereas expression descended to 10.8%
± 1.20%
(p=0.04) and 0.16% ± 0.05% (p<0.001) for IFNAR1 and IFNAR2,
respectively in
day 15 trophoblasts. Gene expression analysis of proapoptotic genes
revealed a
significant increase for XAF1 (4.2- and 22.4-fold at days 15 and 18,
p<0.001, respectively) and TRAIL (11.8-fold at day 18, p=0.006) in
pregnant
animals. However, in day 18 trophoblasts XAF1 transcript levels only
accounted
for 0.003 % ± 0.001% when compared to gravid endometrium at day
18. For the
XAF1 antagonist XIAP and for other antiapoptotic genes (Survivin,
BclxL, FLIP)
tested no significant changes were detected in the endometrium.
Remarkably,
next to abundant Survivin transcripts in day 15 trophoblasts (157-fold
higher
than day 15 endometrium, p=0.002) we detected an 82-fold increase of
FasL
tanscripts comparing day 15 to day 18 trophoblasts. If secreted, FasL
could
therefore act as additional proapototic ligand on the endometrium.
However, a
colorimetric TUNEL assay showed no increase in apoptotic cells numbers
in the
endometrium. Furthermore, expression analysis of effector caspases 3
and 7
revealed no modification in the endometrium throughout cycle and early
gravidity which was consistent with luminometrically caspase activity
assays in
tissue homogenates. Although bovine concepti showed reduced IFN
receptor expression
there might be IFN-independent mechanisms for the initiation of
apoptotic
pathways in bovine embryos at the transcript level. P086 Effects
of Masson Pine pollen extracts on the gene expression profile of
porcine ileal
cell cultures 1Institute
of Physiology, Centre of Life and Food Sciences (ZIEL), Technical
University of
Munich; 2Institute Biopolymer Chemistry, Technical
University of
Munich; masanetz@wzw.tum.de Masson Pine
pollen has been used in
the traditional Chinese medicine for several hundred years and it is
said to
have health promoting effects. Its main application has been the
treatment of
disorders of the digestive system but pine pollen has also been used
for a
variety of other medicinal or cosmetic purposes. But only in recent
years some
evidence has been found that pine pollen and its compounds do have
effects e.g.
on inflammatory activities in mice or on TNFα expression in piglets and
at
least a part of these effects have been attributed to the content of
polyphenols in pollen. In the
present study different
extracts of Pinus massoniana pollen were analyzed for their
effects on
cell proliferation and mRNA expression levels of selected genes. Cell
proliferation was analyzed using an electronic cell impedance sensing
technique
and relative gene expression profiles were investigated using qRT-PCR.
It was
found that water and 50% ethanol extracts of Masson Pine pollen in a
concentration equivalent to 1% unprocessed pollen decreased cell
proliferation
significantly (p<0.5) while 100% ethanol, 80% methanol and hexane
extracts
had no effects on cell proliferation. At the same concentration only
the 50%
ethanol extract led to a significant up-regulation of the relative
expression
levels of the pro-inflammatory genes IL6 and IL 8 and to a
down-regulation of
proliferation promoter Cyclin A (p<0.05). LC-ESI-MS was
performed in order to
characterize the compounds responsible for these effects and a number
of
distinct mass signals has been identified that can be found in the 50%
ethanol
extract but not in the aqueous extract. A detailed identification of
these
substances was not yet possible due to missing reference substances –
with the
exception of floretin and naringenin. P087 1Institute
of Marine Research, An RNAi
experiment knocking down a
hormone receptor in the salmon louse ( Lepeophtheirus salmonis )
was
carried out and the effects were evaluated in the next generation.
Hatching
success and phenotypical abnormality in the larvae was investigated.
Q-PCR was
performed with RNA from the mother louse to address the knock down
efficiency
of the hormone receptor following the RNAi experiment. Hormone
receptors do not necessarily
have to be highly expressed as they are switches in the beginning of
gene
cascades. Therefore it is not astonishing that there could be seen very
big
effects in the development of the larvae even though little down
regulation was
found. Two different analytical methods (the ∆∆Ct method and a kinetic
approach) were compared and the results were discussed serving as an
example of
the importance of choosing the appropriate methods when evaluating
Q-PCR data. P088 Investigation
of the potential transfer of recombinant DNA from feed into milk of
cows fed
genetically modified maize 1Technische Despite the
consecutive increase in
global adoption of genetically modified (GM) crops, there is an ongoing
debate
about potential effects and the fate of recombinant DNA in the food
derived
from animals fed GM crops. To address the food safety concerns of the
public
regarding the potential transfer of recombinant DNA ( cry1Ab )
into the
milk of cows fed GM maize (MON810), a highly specific and sensitive
qPCR assay
was developed for monitoring suspicious presence of novel DNA in bovine
milk.
The developed assay was validated according to the “Minimum Performance
Requirements for Analytical Methods of GMO Testing” published by the
European
Network of GMO Laboratories (ENGL). 36 lactating
Simmental cows, housed
at the DNA from
whole milk was isolated
following the optimized procedures published by the Federal Office of
Public
Health (FOPH, The detection
limit of the qPCR was
100 copies of cry1Ab per µL milk. A mean recovery rate
of 84.9% (n=3,
six replicates each), an intra-assay CV of 0.15 (n=9) and an
inter-assay CV of
0,78 (n=9, three replicates each) illustrate the suitability of the
extraction
and quantification procedure for novel DNA in whole milk. Using this
assay for
milk samples collected from cows fed either transgenic or
non-transgenic
rations for 25 months, fragments of cry1Ab were not detected
in the
analyzed samples at the assay detection limit. P089 1Institute
for Agricultural and Fisheries Research, Burg. van Gansberghelaan 96,
9820 C. acutatum is
one of the most important fungal pathogens of strawberry worldwide. The
disease
is responsible for up to 80% plant death in nurseries and yield losses
of over
50% in strawberry production fields. Symptoms include fruit rot, crown
rot and
lesions on stolons. However, C. acutatum may also persist on
young
strawberry plants without causing visible symptoms. Such latent
infections are
considered to be the main cause of dissemination of C. acutatum .
Detection and quantification of C. acutatum during this latent
phase
using real-time PCR might aid considerably in the reduction of its
spread. This
paper describes the development of real-time PCR assays using primers
and
probes designed to the multi-copy rDNA ITS1 region and the single-copy
β-tubulin
2 gene of C. acutatum . The sensitivity of both assays is
compared and
the data are used to calculate the genome size and ITS copy number of C.
acutatum . Finally, detection and quantification of C.
acutatum is
demonstrated in artificially and naturally infected strawberry leaves. Using TaqMan
technology, the
ITS-based real-time PCR assay could reliably detect as little as 50 fg
genomic
DNA, 100 copies target DNA, or 25 conidia. The β-tubulin-based assay
was circa
66 times less sensitive, and therefore less suitable for detection
purposes.
However, by applying the β-tubulin primers together with the ITS
primers to
both genomic DNA and known numbers of cloned target DNA, they proved
very
useful in revealing some insights into the genome of C. acutatum .
Specifically, we estimated a genome size of 60.0 Mbp for C.
acutatum and
the presence of circa 20 tandem copies of the ITS region in one genome
of C.
acutatum . Concerning the detection of C. acutatum in
strawberry
leaves, we were able to detect C. acutatum in plant tissue
mixes
containing only 0.001% infected tissue. In addition, real-time PCR
analysis of
leaf samples taken at various times after inoculation indicated that
the assay
allows monitoring of early symptomless growth of C. acutatum on
strawberry
leaves. Finally, the assay allowed detection of C. acutatum in
naturally
infected and symptomless strawberry leaves collected from production
fields and
planting material. In
conclusion, our results
demonstrate that the ITS-based real-time PCR assay developed in the
present
study, is a highly sensitive technique that can be used in routine
quarantine
inspections to screen strawberry planting material for C. acutatum contamination. P090 The
use of TaqMan qPCR to detect poultry ingredient in white fish surimi 1Kimron
Veterinary Institute, Surimi, a
minced and washed fish
muscle products consisting of salt soluble-myofibrillar proteins, is
used as an
functional ingredient in seafood analogs. The most common fish used in
the
production of surimi is Alaska Pollock or white fish ( Theragra
chalcogramma ). One reason white fish is so widely used in the
production
of surimi,
besides its abundance, is the yield of cohesive gels created during
processing,
which is essential for the creation of the final product. Surimi is
mainly produced in the
Far-East as a source of alternative fish protein. In produce of this
sort there
is always a chance of proteins other than fish, being incorporated such
as
proteins of poultry origin with the danger of introducing poultry that
was
exposed to the highly pathogenic avian influenza virus. In an effort
to develop a system
that will enable detection of proteins of poultry origin in imported
surimi, a
TaqMan real-time qPCR reaction which was specifically designed to
detect
chicken mitochondrial cytochrome c sequences, was implemented. To examine
the reaction specificity,
white fish, chicken embryo, chicken breast, pig kidney tissue culture
cells and
cattle mitochondrial DNAs were tested. Only
mitochondrial DNAs extracted
from chicken embryo and breast, reacted positively, contrary to
negative
reactions when mitochondrial DNAs from white fish, pig kidney tissue
culture
cells and cattle meat were used. To test the
presence of materials of
poultry origin in surimi, mitochondrial DNA from surimi was extracted
and
served as template in the reaction. Positive reaction in the surimi
samples
indicating the presence of chicken origin materials could be
demonstrated. P091 UTILIZATION
OF FTA (R) CARDS COMBINED WITH ONE-STEP REAL-TIME PCR FOR RAPID
DETECTION OF
QUARANTINE VIRUSES AND PHYTOPLASMAS Institute for
Agricultural and
Fisheries Smooth
international trade of planting
material often depends on efficient inspections at the port of entry.
Therefore, reliable and fast screening for regulated plant pathogens is
an
important challenge. This study evaluates the use of Whatman FTA (R)
card
technology (a commercially available filter paper impregnated with
patented
chemicals) as RNA and DNA extraction method compared to commercial
plant
extraction kits (DNeasy, RNeasy Plant – Qiagen; Invisorb DNA and RNA
Plant –
Invitek), combined by a one-step qPCR detection for a selection of
regulated
pathogens in host plants. The method was tested for 2 viruses ( Pepino
mosaic virus , Potexvirus, Flexiviridae; Tomato spotted wilt
virus ,
Tospovirus, Bunyavirudae) and a phytoplasma ( Apple proliferation
phytoplasm , AP Phytoplasma). A dilution series of respective in
vitro transcripts
of the target RNA fragment served as standard for quantification of the
RNA
viruses in the infected leaf samples. Quantification of phytoplasma DNA
was
done by means of plasmids containing a 16S rDNA target fragment. Both
were
added to a mock-RNA/DNA extraction of healthy control plants. With the
commercial RNA and DNA extraction kits detection was more sensitive and
quantification more reliable than with the FTA card method. However,
the FTA
card technology followed by a one-step qPCR detection enabled reliable
screening of leaf tissue for infection with our target regulated
pathogens in
less than 4 hours. For the viruses, the RNA fixation on the filter
paper was
most efficient when the leaf samples were homogenised in a standard
ELISA
extraction buffer. For AP Phytoplasma DNA, a direct leaf punch on the
FTA cards
was sufficient for a reliable detection of this pathogen in infected
leaf
tissue. In conclusion we can state that the FTA card technology
followed by
one-step qPCR could offer a fast and reliable screening method for
sanitary
control on the regulated pathogens. P092 1Technische
Universitaet Muenchen, Germany; 2Bayrische Landesanstalt
für
Landwirtschaft, Institut für Tierzucht, Grub, Germany; 3Klinikum
der
Johann Wolfgang Goethe-Universität Frankfurt, Pharmazentrum
Frankfurt/ZAFES,
Institut für Klinische Pharmakologie, Frankfurt, Germany; ulbrich@wzw.tum.de The
establishment of pregnancy is
dependent on an intact embryo-maternal communication especially prior
to
implantation. Prostaglandins (PG) are important regulators of several
reproductive processes. We analyzed the most relevant PG in bovine
uteri at
different preimplantation pregnancy stages as compared to non-pregnant
controls. Additionally, endometrium and trophoblast tissue samples were
examined regarding the expression of specific enzymes and receptors
involved in
PG generation and function. Simmental heifers were artificially
inseminated
(pregnancy) or received seminal plasma only (controls). At days 12, 15
or 18
post estrus uteri were flushed with 100mL PBS for PG determination by
LC-MS/MS.
Endometrial and trophoblast tissue was sampled for RNA extraction and
RT-qPCR
analysis. At all days and time points examined, the concentration of
6-keto-PGF1alpha (PGI2-metabolite) was predominant followed by
PGF2alpha >
PGE2 > PGD2 = TXB2. At days 15 and 18 all PG increased from low
levels at
day 12, with a much higher increase during pregnancy. The highest PG
concentration was measured at day 15 of pregnancy with 6-ketoPGF1alpha
(6.4
ng/mL) followed by PGF2alpha (1.1 ng/mL) and PGE2 (0.3 ng/mL). The
ratio of
PGE2/PGF2alha was neither influenced by day (P=0.09) nor status
(P=0.4). The PG
contribution of the embryo was high as seen from abundant endometrial
PG
synthase transcripts together with low catabolic enzyme expression and
evidenced by higher levels of PG than respective metabolites. While the
endometrium expressed more PGE2 receptor (EP2) than the embryo, the
embryo
revealed more transcripts for PGF2alpha receptor (FP) than the
endometrium.
This study provides comprehensive quantitative insights into the
dynamic regulation
of PG of the bovine uterine lumen in vivo. Next to PGE2 other PG,
namely PGI2
and PGF2alpha may have an important role for the developing embryo and
thus may
be essential rather than detrimental for successful reproduction. Supported by
DFG (Ul 350/1-2, FOR
478) Diagnostic &
Molecular Markers (human)
P093 IDENTIFICATION
AND QUANTIFICATION OF THE RARE BCR-ABL TRANSCRIPTS e13a3 AND e1a3. University OBJECTIVE:
The vast majority of
cases of chronic myelogenous leukemia, and a minority of acute
lymphoblastic
leukemia cases, are associated with the presence of the
Philadelphia-chromosome. This represents a balanced translocation
fusing the
c-ABL gene of chromosome 9 to the BCR-gene of chromosome 22. Most
common
isoforms of BCR-ABL are e13a2 and e1a2, both with fusion of BCR to ABL
exon 2
We, in this poster, describe the detection of two rare BCR-ABL fusion
gene
transcripts, and the establishment of the quantitative analysis for
these
transcripts. METHODOLOGY:
Peripheral blood
samples from two patients were referred to our routine laboratory for
detection
and eventual identification of BCR-ABL fusion gene transcripts. The
first case
is a 59 years old woman with acute lymphoblastic leukemia (ALL). The
second
case is a 26 years old woman diagnosed with chronic myeloid leukemia
(CML). For
determination of BCR-ABL fusion
gene transcripts, two different PCR strategies were used. One was a
qualitative
analysis making use of conventional multiplex PCR. Agarose gel
electrophoresis
of the PCR-products was performed. Simultaneously a real time
quantitative
RT-PCR analysis (RQ-PCR) was conducted with accordance to the
recommendation of
the EAC consortium. The amplicons of interest were sequenced, and the
selected
primers and probes were tested for a Taqman based RQ-PCR. Cloning of
the
patient sample with TOPO TA cloning kit was done to produce plasmids
for the
generation of standard curves. RESULTS: The
amplified products of
the multiplex PCR showed bands with unexpected sizes for both of the
patients.
The PCR product from the patient with ALL had a size of about 230 bp
and that
of the CML patient about 260 bp. The sequencing results showed that the
PCR
product of the ALL patient had an exact length of 235 bp, and
identified the
transcript as a product of an e1a3 fusion gene. Similarly, sequencing
of the
PCR product from the CML patient identified a segment of 266 base
pairs. The
transcript was a product of an e13a3 fusion. By combining the EAC
forward
primers used for detection of e13/14a2 and e1a2 transcript with the ABL
control
gene probe and the reverse primer which are located in exon 3, a RQ-PCR
analysis could be performed. Quantitation of e13a3 was done at
diagnosis and
after the patient had undergone treatment. After 3 months on standard
treatment
(Imatinib mesylate 400 mg once daily) our RQ-PCR showed a 2-log
reduction of
BCR-ABL. Quantification of e1a3 after treatment was not possible as the
patient
died short time after initiation of therapy. CONCLUSION:
Detection of the BCR-ABL
transcripts lacking exon 2 in our multiplex PCR, was made possible due
to the
location of the reverse primer in exon 3 of the ABL-gene. We describe
the
establishing of a real time quantitative PCR analysis, based on the EAC
protocol, which allows the quantification of diagnosis and follow-up
samples of
the rare transcripts e13a3 and e1a3. P094 Somatostatin
and dopamine receptor expression influences the effects of selective
ligands on
cell viability in non functioning pituitary adenomas in vitro Previous
studies demonstrated that
drugs interacting with dopamine (DR) and somatostatin receptors (SSTR)
might be
useful in non-functioning pituitary adenoma (NFPA) medical therapy. The
aim of
our study is to evaluate the effects on cell viability of the SSTR/DR
ligands
BIM23A370 and BIM23A387 (SSTR2>DRD2) in 6 NFPAs in vitro (#1 - #6)
and
BIM23A760 (SSTR2>>DRD2>SSTR5) in 8 NFPAs in vitro (#7 - #14),
investigating SSTR (subtypes 2 and 5) and DR (subtype 2) expression
pattern. Primary
culture cell viability is assessed after a 48 h treatment with each
compound at
10 nM by a colorimetric method. Since the normal counterpart is not
available,
Absolute Quantitative Real Time PCR method is used to quantify SSTR2,
SSTR5,
and DRD2 expression, with a 6 points standard curve (range: 10^3-10^8
molecules
of target cDNA). To normalize the results, each sample is quantified
for
housekeeping gene 18S expression. All the data are expressed as target
cDNA
molecule number/1 µg of retrotranscribed total RNA. SSTR2 is
expressed in 10
NFPAs (71.4 %), at variable levels (6000-720000). SSTR5 is poorly
expressed
(10681) in only 1 NFPA (7.1%), while DRD2 is highly expressed
(51000-2721320)
in 9 NFPAs (64.3%). In samples #1, #4, and #6 expressing SSTR2 but not
DRD2,
treatment with BIM23A370 or BIM23A387 increases cell viability
(15-360%,
p<0.01). In sample #2, expressing DRD2 (51080) but not SSTR2,
treatment with
BIM23A387 induces a 25% (p<0.01) decrease of cell viability, while
treatment
with BIM23A370 doesn’t influence cell viability. In sample #3,
expressing all 3
receptors (SSTR2:144208; SSTR5:10681; DRD2:229926), treatment with
BIM23A370
causes a 12 % (p<0.01) reduction in cell viability, while no change
is
recorded after treatment with BIM23A387. In sample #5, lacking the
expression
of all 3 receptors, treatment with the chimeric molecules results in a
20 %
(p<0.01) increase in cell viability. Treatment with BIM23A760 does
not
influence cell viability in samples #7 - #14, except in sample #8,
expressing
DRD2 (73494) and SSTR2 (114705), where a reduction in cell viability
(-15 %,
p<0.01) is evident. It might be relevant that this sample expresses
SSTR2 at
higher levels than all other NFPAs. SSTR/DR ligand BIM23A370 causes a
significant reduction in cell viability only in NFPA with both SSTR2
and DRD2
receptors reaching an expression level >100000 and >200000,
respectively.
In all other samples, treatment with this chimeric molecule leads to an
increase in cell viability. Treatment with receptor ligand BIM23A387
causes a
reduction in cell viability in samples expressing DRD2 <100000,
while it has
no effect in samples expressing DRD2 >100000. SSTR/DR ligand
BIM23A760
reduces cell viability only in one case showing DRD2
expression<100000 and
SSTR2 expression>100000. In conclusion, these molecules could be
useful in
NFPAs medical therapy, but further investigations are necessary to
assess
SSTR/DR expression pattern in order to maximize effects on cell
viability. P095 VAP
(Ventilator-Associated Pneumonia) DIAGNOSIS USING MOLECULAR MARKERS.
PRELIMINARY RESULTS. HOSPITAL
UNIVERSITARIO CENTRAL DE
ASTURIAS, VAP
(Ventilator-Associated
Pneumonia) is one of the most common infectious complications in
intensive care
units as a consequence of intubation and mechanical ventilation
support. Early-VAP
appears during the first four days of mechanical ventilation while
Late-VAP
develops ≥ five days after. Taking into account that about half of
episodes of
VAP appear, within the first four days of intubation, the possibility
of
detecting the cited increase few days before the apparition of the
symptoms
give us the chance of avoid the progression of the infection. According
with
previous reports using micro array technique several genes related with
immune
activity and immune response to bacteria or infection, were tested in
order to
establish the best marker to the early detection of VAP. Objectives
: Determination of the appropriate marker for the early diagnose of VAP
in
Intensive Care Unit patients mechanically ventilated in order to avoid
the infection
progress. Methods
: Twelve patients (10 men-2 women; mean age
57.58) were included in the preliminary study after being mechanically
ventilated. Patients who develop VAP were included in the problem
group.
Arterial blood samples were obtained each 72 hours since intubation
day,
considered the initial sampling point. Samples were processed to
extract RNA
from leukocyte fraction, cDNA was then synthesised using random
primers.
Afterwards, several genes related with infectious response were tested
by Real
time PCR: Lactotransferrine (LTF), Cathelicidin Antimicrobial Peptide
(CAMP),
Phospholipid Scramblase 1 (PLSCR1), Inhibin Beta A (INHBA) and
Hydroxyprostaglandin Dehydrogenase 15-NAD (HPGD). Results
: After performing relative quantitation at different time points using
as
reference gene β Actine, a marked increase in HPGD expression
(ratio=10.23)
were observed in the problem group versus control at point 3, on the
other hand
the ratio at point 2 (before the apparition of the symptoms) is higher
in
control group than in problem group. Conclusion : HPGD gene
seems to be the most
adequate marker for the early diagnose of VAP in ICU patients with
mechanic
ventilation. Currently, the aim of the present project is to determine
the
adequate time point where the expression increase appears. P096 Novel
Protein Expression Assays using qPCR for the Detection and Relative
Quantification of Protein Markers in Human Embryonic Stem Cells 1Applied
Biosystems, part of Life Technologies, Germany; 2Applied
Biosystems,
part of Life Technologies, Foster City, USA; 3Applied
Biosystems,
part of Life Technologies, United Kingdom; Astrid.Ferlinz@eur.appliedbiosystems.com
Quantitative
PCR (qPCR) has
revolutionized the characterization of nucleic acids in cells, and
several
classes of cellular nucleic acids are routinely analyzed by qPCR
assays,
including genomic DNA, mRNA and microRNAs. Proximity ligation assay
(PLA) technology
extends qPCR applications now to the detection of cellular proteins
through the
amplification of a surrogate DNA template. PLA is a three-step process
that
involves, 1) binding of paired antibody-oligonucleotide probes to a
protein
target in biological samples, 2) templated ligation of the
oligonucleotides in
proximity, and 3) qPCR detection. We have optimized this technique for
crude
cell lysates utilizing a simple, one-step sample lysis approach to
release all
classes of proteins, and combined it with gold standard TaqMan®
chemistry to
create a highly sensitive and specific process for measuring protein
expression
in small samples. One application of this assay is the detection and
relative
quantification of markers in pluripotent and differentiated stem cells.
Stem
cell characterization typically relies on determining the presence and
amount
of stage specific protein markers such as OCT4, NANOG, SOX2, and LIN28.
Protein
expression results confirm cell-stage specific changes in protein
expression of
these key stem cell markers, and the data can be directly compared with
published mRNA expression profiles for the same cell lines. We have
engaged a
number of researchers as test sites for these assays and are gathering
input
and feedback. Our findings illustrate how this new assay system expands
the
scope of qPCR to protein detection and quantification, an important
area of
cell biology. P097 The
developmental changes in mitochondrial DNA content and expression
levels of
genes involved in pathway from PGC1 to mtDNA replication in human
tissues The
inadequate efficiency of
mitochondrial biogenesis leads to low energy production which may play
a
crucial role both in the fetal development and neonatal morbidity.
Therefore we
focused our work on finding the first markers of mitochondrial
proliferation on
transcriptional level during human prenatal development. The aim of our
study
was to characterize the changes in expression of genes involved in the
regulation and maintenance of mitochondrial DNA content (peroxisome
proliferatoractivated
receptor-g coactivator-1a, PGC1; nuclear respiratory factor 1, NFR1;
mitochondrial transcription factor A TFAM; polymerase gamma – catalytic
subunit, POLG). Presently we analyzed both mtDNA amount and expression
levels
of chosen genes in human fetal tissues during gestation. DNA and RNA
were
isolated from 26 pairs of liver and muscle tissue samples obtained at
autopsy
from miscarriages after informed consent, between 13 th and 28th week
of
gestation. The mtDNA amount and gene expression levels were analyzed by
the
real-time PCR method using SybrGreen I. In both fetal liver and muscle
tissues,
mtDNA content and TFAM expression levels were increasing with onward
fetal
development (mtDNA: r = 0,50; p < 0,01; respectively r = 0,62; p
< 0,01);
(TFAM: r = 0,56; p < 0,01; respectively r = 0,61; p < 0,01). On
the other
hand, POLG expression level was increasing only in fetal liver tissue (
r =
0,54; p < 0,01). NRF1 was unchanged in both fetal tissues and PGC1
was
slightly rising between 13 th and 28th week of gestation in liver
tissue (r =
0,42; p < 0,05). Our results showed that POLG expression varies
between two
different tissues during gestation and probably is not associated with
mtDNA
content as tightly as TFAM expression. The increase of PGC1 transcript
level is
statistically significant in liver tissue and we suggest that it could
bear
evidence of tissue specific expression or regulation. Therefore the
binding of
NRF1 to its specific sites which are situated on the promoter of TFAM
is
positively affected by increasing expression of PGC1 in liver tissue. In
conclusion, this study described
mainly the increasing trends of gene expression in the pathway leading
from
expression of PGC1 to mtDNA content changes in fetal liver and muscle
tissues
during second trimester of gestation. Our study involved the largest
set of
human fetal tissue samples as it has been used for such study to date.
According to our results, we suppose that the mitochondrial
proliferation is
growing up between 13 th and 28th week of gestation. Therewithal it is
the
first hallmark of prepare for postnatal adaptation which is evident on
transcriptional level in this fetal period. This work was
supported by grant
GAUK25755707, IGAMZ-NR9410,GACR305/08/H037. Target-assembled
in-situ detection by DNA-mounted exciplexes, heteroexcimers and
excimers based
on 2-arylalkynyl-pyrene. National
Medical Research Centre; abdulgbaj1@hotmail.com
Target-assembled
in-situ detection
by DNA-mounted exciplexes, heteroexcimers and excimers based on
2-arylalkynyl-pyrene. Reversible
hybridisation of
complementary polynucleotides is essential to the biological processes
of
replication, transcription, and translation. Physical studies of
nucleic acid
hybridisation are required for understanding these biological processes
at a
molecular level. In addition the physical characterisation of nucleic
acid
hybridisation is essential for predicting the performance of nucleic
acids in
vitro, for instance, in hybridisation assays used to detect specific
polynucleotide sequences. In the
recently discovered
split-oligonucleotide (tandem) probe system for exciplex-based
fluorescence
detection of DNA, the detection system is split at a molecular level
into
signal-silent components. For fluorescence emission by the
exciplex-forming
system of two short probe oligonucleotides and their target nucleic
acid, the
components must be assembled correctly and very precisely into a
specific
3-dimensional structure to ensure close proximity of the exciplex
partners. The
study reports the effects of structural variation of the exciplex
partners to
make use of the spectroscopic properties of alkynyl-substituted pyrenes
as
exciplex partners, a structural change which allows excitation to occur
in the
visible region. The exciplexes formed emitted at 505 nm (broad bands)
with
large Stokes shifts (>100 nm). The effects of linker length and
relative
locations of the exciplex partners were assessed for target sequences
based on
(i) a region of the Leishmania genome and (ii) the region corresponding
to the
cytochrome P450 CYP2C9 3*allele (wild type and SNP). The molecular
nature of
the gap region in the tandem nucleotide system constructed for such
tandem
duplexes was studied using melting temperatures based on both the
absorbance at
260 nm and on the fluorescence of the exciplexes formed, as well as by
the
efficiency of the exciplex formed by such systems, the exciplex
providing a new
and extremely sensitive probe of subtle structural and conformational
manifestations in DNA duplexes. Exciplex structural variation provides
a
broader range of structural insight than can be expected from analogous
systems
based on excimers for which the choice of partners is very limited. P099 Nucleic
acid diagnostic tests for the detection of microbial pathogens National The Molecular
Diagnostics Research
Group (MDRG) at NUI Galway has over 20 years experience in the field of
molecular diagnostics. Research activities include molecular diagnostic
target
discovery and nucleic acid based test design and development. Over the
years
the group has identified a number of platform nucleic acid diagnostic
test
(NAT) target technologies for the sensitive and specific detection and
identification of bacterial and fungal pathogens. The NAT target
technologies
have been demonstrated in a wide range of test formats including,
real-time
PCR, real-time NASBA and direct nucleic acid target detection systems.
Applications of the NAT technology include clinical, food, veterinary
and
environmental sectors. Successful commercialisation of our nucleic acid
targets
has occurred through licensing agreements and research and development
collaborations with many of the major international diagnostics
companies. Detailed
below are some of the strategies and methodologies employed by the MDRG
in the
design and development of NATs for the detection and identification of
micro-organisms. D1
– D5 dopamine receptors expression in paranoid schizophrenia patients Islamic azad
university of Schizophrenia,
commonly developed in
young adults that is one of most common mental disorders, but the
phatophysiology and etiology conditions of schizophrenia is still
obscure.
Based on Numerous studies about dopamine and schizophrenia, it
suggested that
changes in the dopamine systems are in related with schizophrenia, but
still
there is no clear direct evidence for dopamine hypothesis in
schizophrenia. In terminated
examination, 30
paranoid schizophrenia patients mRNA from white blood cells extracted ,
then
cDNA were synthesized . After Quantitive Real-time PCR examination with
the
related primaries for D1 – D5 receptors were terminated and the
compared
consequences in abundance of genes expression with the normal samples
reveals
that D1 – D5 dopamine receptors were expressed in all samples .
Abundance of
normal individuals were D1 100% , D2 16.6 % , D3 40 % , D4 83.3 % , D5
86.6 %
and for patients were D1 100% , D2 83.3 % , D3 30 % , D4 83.3 % , D5
83.3 % . P101 1Department
of Laboratory Medicine, Background and aim Celiac
disease (CD) is an
inflammatory disease, triggered by an immunologic response to gluten in
wheat,
which manifests itself by symptoms such as diarrhoea, abdominal pain
and
malabsorption. CD results in an inflamed small intestine with varying
degree of
villous atrophy, crypt hyperplasia and increased paracellular
permeability of
the intestinal epithelium. According to current guidelines, the
diagnosis of CD
requires intestinal biopsy and is set by histopathological findings.
The aim of
this study was to investigate the possibility of establishing molecular
classifiers using gene expression data. Materials and methods Unselected
paediatric
patients investigated for suspected CD were consecutively included in
the
study, and total RNA was isolated from small intestinal biopsy samples.
Using
real-time PCR and relative quantification, gene expression was
investigated in
biopsies from four healthy individuals, ten with CD
(histopathologically Marsh
1 – 3C) and one biopsy obtained after introduction of gluten-free diet.
A total
of 33 genes were analysed: five villi markers, 11 crypt markers and 17
apical
junctional complex (AJC) genes. Expression data was normalized using
two
reference genes exhibiting low sample-to-sample variation (M-value of
0.19
according to the geNorm algorithm), and statistically investigated for
differential expression using Mann-Whitney (p < 0.05, uncorrected
for
multiple testing). Sample classification based on gene expression
profiling was
explored by means of principal component analysis (PCA) and
hierarchical
clustering. The study was conducted under approval by the regional
ethics
committee. Results Sixteen of
the investigated genes
were differentially expressed (healthy vs. Marsh grade 2-3): all of the
villi
markers, three crypt markers and eight AJC genes. All of the
differentially
expressed genes except two of the crypt markers (0.63- and 0.72-fold in
healthy
compared to Marsh grade 3) exhibited higher expression (1.3 – 3.5-fold)
in
healthy biopsies. Based on the degree of intestinal mucosal lesion
classified
according to Marsh criteria, PCA and cluster analysis resulted in the
following
three relevant groups: healthy / Marsh type 0 / Marsh type 1, Marsh
type 2, and
Marsh type 3. Conclusions Preliminary
data indicate that gene
expression profiling of intestinal biopsies is a feasible approach to
classification and that this may prove useful in confirming the
diagnosis. Identification
of Bacillus anthracis by a specific chromosomal marker 1Bundeswehr
Institute of Microbiology, Munich, Germany; 2Bavarian
Agency for
Health and Food Safety, Oberschleissheim, Germany; 3Health
Protection Agency, Porton Down, Salisbury, Wiltshire, United Kingdom; markusantwerpen@bundeswehr.org
Correct
identification of Bacillus
(B.) anthracis and distinguishing this organism from closely
related B.
cereus and B. thuringiensis is still one of the big
challenges in B.
anthracis diagnostics. The differentiation between non-anthracis Bacillus
-species harbouring anthrax-specific virulence plasmids,
plasmidless and
plasmid-harbouring B. anthracis strains is one of the major
problems of commercially
available PCR-kits. Primer and probes for amplification of chromosomal
markers
are either not present or not specific. Many markers have been
describedto be
highly specific, but overtime, several exceptions for these markers
became
public. Today, analysis of published genomes reveals possible marker
genes to
be more specific for B. anthracis , like the locus BA_5345. In
this
study we designed a TaqMan® PCR targeting sequence of the latter
gene. A panel
of 328 Bacillus species was used to determine specificity.
Probit
analysis was performed to ascertain sensitivity. All B. anthracis isolates
(n=92) were specifically detected by using the genomic TaqMan® PCR
assay
whereas 236 strains belonging to 19 Bacillus species other
than B.
anthracis showed negative results. The detection limit was
calculated to be
12.7 copies per reaction (95% confidence interval, 10.2 – 17.5 copies).
Here we
present a well-evaluated and actually highly specific TaqMan® PCR
assay for the
unequivocal detection of B. anthracis based on a chromosomal
marker. P103 Quantitative
real-time PCR measurement of NPM1A Mutations following Allogeneic Stem
Cell
Transplantation in Patients with Acute Myeloid Leukemia University
Medical Centre Background:
Following allogeneic
stem cell transplantation, minimal residual disease (MRD) diagnostics
in acute
myeloid leukemia (AML) provides an option for early detection of
relapse. The
Nucleophosmin (NPM1) mutations represent the so far most frequent known
molecular marker in AML with a specific association to normal karyotype
AML.
Most frequent is the NPM1 subtype consisting of a four base pair
insertion. We
here evaluated the utility of this marker for follow-up in the
post-transplant
period in AML. Patients and Methods: A cohort of 13 mutated AML
patients
receiving 14 allogenetic stem cell transplantations (SCT) from related
or
unrelated donors was retrospectively screened by quantitative real-time
PCR
(qPCR) for NPM1A mutations on bone marrow or peripheral blood samples
which had
been cryopreserved before and after SCT. Normalization was performed by
quantification of the HCK gene. Median follow-up was 7 months (range, 1
– 60
months) from SCT. Results: After SCT, 10 of 14 mutated transplantation
cases
(71%) became PCR-negative. Four of those achieved stable remission. In
contrast, all four cases (29%) remaining NPM1Amut positive
post-transplant
developed morphological relapse, in all being accompanied/preceded by
increase
of the NPM1A/HCK mutation level. Increase of NPM1mut was seen earlier
than
morphological relapse with a mean interval of 24 days (range, 12-38
days).
Conclusions: According to this series, quantitative assessment of
NPM1mut seems
suitable to follow MRD in the post-transplant period and can indicate
relapse
earlier than morphology. This might allow an earlier start of adoptive
immunotherapy in case of impending relapse. P104 Bladder
cancer detection with survivin mRNA as a molecular marker 1BGFA-
Research Institute of Occupational Medicine, German Social Accident
Insurance,
Ruhr-University Bochum, Germany; 2Department of Urology,
Eberhard
Karls Universität Tuebingen, Germany; bontrup@bgfa.de Objective - The presented
investigation was
carried out in patients with suspected bladder tumors to evaluate the
feasibility and discrimination characteristics of survivin mRNA
measurement as
a diagnostic and potential prognostic molecular marker for transitional
cell
carcinoma (TCC). Aim of the study was to reveal possible influencing
factors
and estimate a cut-off for the discrimination between positive and
negative
diagnostic findings. Methods - Survivin was
measured by an mRNA assay with anonymized cell samples isolated from
voided
urine. The specimens were taken from 50 patients with the suspicion of
new or
recurrent TCC prior to transurethral resection. The study group
consisted of 33
male and 17 female patients. Mean age was 68 years. Histopathological
evaluation revealed no malignancy in 18 (36%) patients and confirmed
TCC in 32
(64%) patients (9 pTaG1, 11 pTaG2, 4 pT1 G2-3, 4 > pT2, 4 pCis). To
preserve
the samples, cell pellets of centrifuged urine samples were frozen in
buffer
solution. These samples were shipped to BGFA laboratory, where RNA was
isolated
and reverse transcribed, before survivin mRNA was amplified by nested
PCR and
quantitated by Real-time PCR. ß-Actin mRNA was determined for
sample quality
control. Furthermore ROC analyses for the survivin assay were performed
considering the influence of hematuria, cystitis, and other urological
diseases. Results - The
determination of ß-actin showed
that 49 (98%) of the samples had sufficient integrity for the mRNA
assay after
freezing and shipping. The ROC analysis identified a cut-off level of
10,000
mRNA copies of survivin, resulting in a sensitivity of 53% and a
specificity of
89%. Six out of the 20 pTa tumors (30%), all pT1 and all invasive
tumors were
detected. Of the 4 patients with pCis 3 (75%) could be identified. Only
two
patients (4%) were assessed as false positive. One of them suffered
from
urolithiasis and the other is under ongoing control because of positive
cytology findings. The different variables as diseases, age, and gender
did not
significantly influence the marker levels. Conclusion - Survivin
might be a suitable
diagnostic marker for bladder cancer. In this study, we found no
confounding
factors of survivin mRNA levels but the statistical power was limited.
The
specificity (89%) was very promising especially considering the
clinical
information about the false positive patients. Sensitivity in
high-grade tumors
was excellent (91%), however, the lower sensitivity on low-grade tumors
needs
to be evaluated in large prospective studies to assess survivin as
screening
marker. P105 REAL-TIME
PCR ANALYSIS OF NNMT EXPRESSION IN URINE AND TUMOR TISSUE OF BLADDER
CANCER
PATIENTS AND ITS POTENTIAL RELEVANCE FOR DISEASE DETECTION. Università
Politecnica delle Carcinoma of
urinary bladder ranks
among the top ten most common cancers worldwide and the majority of
these
tumours recur and progress into muscle-invasive disease, thus needing
for
long-term and frequent follow-up. Despite its invasive nature,
cystoscopy
remains the gold standard diagnostic evaluation for detection and
surveillance
of bladder cancer. Urinary cytology is used as an adjunct to this
procedure,
but lacks sensitivity for low-grade tumours. More sensitive and non
invasive
methods are therefore required, what fosters continuing interest in
identifying
new bladder cancer markers. To explore
the involvement of
enzymes of drug transport, phase I metabolism, and phase II metabolism
in
bladder cancer, in the present study we analysed the gene expression
profiles
of tumour and non-tumour tissues obtained from the same patient by DNA
macroarray. The enzyme Nicotinamide N-methyltransferase (NNMT) 1 was
identified
as a highly expressed gene in bladder cancer. Real-Time PCR, Western
blot
analysis, and catalytic activity assay performed on a large cohort of
patients
with transitional cell carcinoma (TCC) confirmed NNMT upregulation.
Differential specific activity measurements (tumour versus adjacent
normal
tissue/ carcinoma in situ) revealed NNMT overexpression in all TCCs,
with fold
change values ranging between 1.3 and 383.2 ( mean 49.4-fold). NNMT mRNA
levels were also
determined in urine specimens obtained from 20 patients with bladder
TCC and 20
healthy subjects. We found that NNMT expression levels were
significantly
higher in patients with bladder tumour compared to controls, that
showed very
low or undetectable amounts of NNMT transcript. Our
experimental data indicate that
a marked NNMT increase is a peculiar feature of TCC and suggest the
potential
suitability of urine NNMT expression levels determination for the
diagnosis of
bladder cancer. 1. Sartini D
et al. (2006) J Urol
176(5): 248-54. P106 Analysis
of the Survival motor neuron gene promoter: effects of histone
deacetylase
inhibitors on histone acetylation, expression, and DNA methylation University
Hospital Brno, Czech
Republic; lfajkusova@fnbrno.cz
Spinal
muscular atrophy (SMA) is
caused by defects in Survival motor neuron (SMN) gene. Two SMN genes
exist in
human genome, a telomeric SMN1 and a centromeric SMN2. Despite their
similarity
in coding sequence, only SMN1 produces sufficient levels of a
full-length mRNA
transcript, while SMN2 is spliced by exon-skipping into non-functional
deletion
variant. In SMA patients SMN1 is disrupted or lost. Recent studies show
a
partial improvement of clinical state of SMA patients upon
administration of
histone deacetylase (HDAC) inhibitors. To investigate molecular
principles
underlying the therapeutic effect of HDAC inhibitors we examine: i) the
effect
of HDAC inhibitors (VPA and M344) on histone acetylation of the SMN
promoter
and SMN2 gene expression; ii) the effect of HDAC inhibitors on BCL2
expression;
and iii) DNA methylation status of SMN promoter and its changes in
response to
VPA treatment. Our results demonstrate, that M344 is more efficient
HDAC
inhibitor compared to VPA, but the histone hyperacetylation induced by
either
of these inhibitors is not sufficient to increase substanially SMN2
expression,
or to shift its splicing pattern towards a full-length variant.
However, up to
4-fold increase was observed in expression of the anti-apoptotic gene
BCL2
after VPA treatment. Further, we evaluated DNA methylation status and
DNA
methylation changes in the SMN2 promoter region after VPA treatment.
The
decrease in methylated CpGs was 6% (from 55 to 49%), thus showing
relatively
weak demethylating effect of VPA, at least in this particular region. The work was
supported by the grants
of MSMT LC06023 and MSM0021622415. P107 Gene
Expression Profile of hOGG1 and its Splice Variants in Human Skin
Samples and
Cell Cultures Human
8-oxoguanine DNA glycosylase 1
(hOGG1) is the most important base-excision repair enzyme to prevent
mutations
by 7,8-dihydro-8-oxoguanine (8-oxoG), the major DNA damage caused by
reactive
oxygen species (ROS). Eight different isoforms caused by alternatively
spliced
mRNAs of hOGG1 are described and classified in two main groups (type 1
and type
2) depending on the last exon of their mRNA sequence. The
characteristics of
any of these isoforms have not yet been determined, but type 1a
(targeted to
the nucleus) and type 2a (targeted to mitochondria) were identified as
the two
main isoforms in several human tissues. In skin hOGG1 plays an
important role
in preventing pathological processes like skin aging and carcinogenesis
caused
by UV-induced ROS. Despite its crucial role it has not yet been
examined which
splice variants of hOGG1 are most prominent in human skin cells and how
they
are regulated. In this study we determined the expression of hOGG1 and
its
splice variants in human skin samples and cultured fibroblasts,
keratinocytes
and melanocytes. Therefore we designed primer pairs for every hOGG1
splice
variant by using different exon combinations respectively exon-exon
transitions
to determine the expression of the splice variants separately. For the
relative
quantification of hOGG1 and its splice variants SYBR-Green qRT-PCR was
used.
The normalization was carried out against either β-Actin to determine
the
overall expression of hOGGI and its correlation to the expression of
single
splice variants in detail, as well as against hOGG1 itself to estimate
the
contribution of the splice variants to the overall expression. The
analyzed
cell types showed no significant differences concerning their splicing
patterns.
Isoform 1b was most expressed, followed by 2e before 2a and 1c. The
isoforms
2b, 2d, 2c and 1a showed only low expression levels. Thereby splice
variants
1b, 2d, 2e, 2a and 2c showed a good correlation (R^2 = 0.6 -0.9) with
hOGG1
expression levels. The splice variants 1a, 1c and 2b were not
correlated with
hOGG1 expression (R^2 = 0.05 -0.2). Also we could identify a very weak
expressed additional hOGG1 splice variant by RT-PCR and subsequent
cloning. The
high expression level of 1b has not yet been detected in other tissues
and
might indicate a special role of this isoform in human skin cells. Also
the
full length nature of hOGG1 1b has not yet been analyzed. It is
putatively
targeted to mitochondria, may be the major mitochondrial targeted hOGG1
isoform
in skin and features therefore a potential suitability as biomarker for
mitochondrial DNA repair. Further experiments are planned to confirm
these
results by testing hOGG1 gene expression in other tissues and cell
types with
known splicing patterns and by overexpression / silencing experiments
of single
hOGG1 isoforms. P108 Quantitative
real-time PCR for the detection of Streptococcus pneumoniae from the
Viridans
group streptococci targeting the cpsA gene Chung-Ang
University College of
Medicine, Korea, South (Republic of); kimwy@cau.ac.kr
Although the
pathological
manifestation may be different, S. pneumoniae shares over 99%
of 16S
rDNA homology with the closely related species, namely S. mitis and
S.
oralis . Therefore, classification of these organisms has long
been
considered difficult. Suppression subtractive hybridisation was
performed to
search for genomic differences between S. pneumoniae and the
most closely
related species S. mitis . About 240 S. pneumoniae -specific
clonal libraries were evaluated with Southern hybridisation and
completely
sequenced. Subsequently, S. pneumoniae -specific primers were
designed
and specificities were examined by gradient PCR using genomic DNAs
extracted
from 132 oral streptococci and other closely related species. Of those
primer
sets, primers based on cpsA gene amplified only the genomic DNAs from
S.
pneumoniae strains. Real-time PCR assays designed for the detection of
specific
sequence regions of cpsA genes were developed (cpsA-rt). The linear
regression
of standard curves for quantitative real-time PCR assay indicated
highly
correlations between the log numbers of S. pneumoniae cells
and the CT
values (R2 = 0.99). The limit of detection was 3 pg purified genomic
DNA,
equivalent to 1000 cells per PCR mixture of a pure culture. These new
oligonucleotide primer set may be very useful for the rapid
identification and
diagnosis to discriminate from other Viridans group streptococci. P109 Augmented
Particle Trapping and Attenuated Inflammation in the Liver by
Protective
Vaccination against Plasmodium chabaudi Malaria 1University
for Verterinary Medicine Hannover, Germany; 2Division of
Molecular
Parasitology and Centre for Biological and Medical Research,
Heinrich-Heine-University Düsseldorf, Germany; 3Max
Planck Institute
for Immunobiology, Freiburg, Germany; mail@juergen-kruecken.net Efforts to
develop a vaccine against
malaria have not been successful yet, reflecting our fragmentary
knowledge
about protective mechanisms. We have analyzed changes in hepatic gene
expression
comparing BALB/c mice succumbing to infection with Plasmodium
chabaudi to
those surviving after vaccination. Mice were vaccinated with host cell
plasma
membranes isolated from P. chabaudi -infected erythrocytes.
Hepatic and
splenic capacity to trap particulate material was determined after
injection of
fluorescent polystyrol beads. Gene expression in the liver was measured
using
real-time RT-PCR. In our vaccination model, survival of BALB/c mice was
raised
from 0% to 80% and peak parasitemia was decreased by about 30%.
Vaccination
boosted particle trapping capacity of the liver during crisis when
splenic
trapping is minimal due to spleen ‘closing’. Real-time RT-PCR revelaed
that
vaccination attenuated malaria-induced inflammation in terms of
expression of the
proinflammatory cytokines IL-1, IL-6, TNFα and the macrophage
activation
markers iNOS and arginase. In contrast, vaccination strongly increased
hepatic
production of IFN-γ which has been associated with protective TH1
immune
responses against P. chabaudi malaria. Moreover, an improvement of
liver
metabolism, which is severely disturbed during malaria infections,
could be
detected by measuring expression of the phase II enzyme Sult2a. In
particular,
vaccination restored inducibility of Sult2a by TCPOBOP, an activator of
the
constitutive androstane receptor. Our data support the view that the
liver is
an essential effector site for a vaccine against blood stage malaria:
vaccination attenuates malaria-induced inflammation thus improving
hepatic
metabolic activity. P110 Investigation
of techniques and applications for rapid detection of α-globin gene
mutation
and deletion 1Beijing
Chaoyang Hospital affiliate of Capital Medical University, China,
Peoples
Republic of; 2Liuzhou women and children’s Hospital,
Liuzhou City,
Guangxi Autonomous Region, China; 3Transgenomics Ltd
(Beijing,
China); 4Beijing Deyi Clinical Diagnostic Laboratory; liujingzhong@hotmail.com Aim: Populations
in Southeast Asia and South China
have high frequencies of α-thalassemia caused by the α-globin gene
mutations
or/and deletions. This study was designed to find an efficient and
simple
diagnostic test for α2-globin gene mutations. Methods : A duplex polymerase chain reaction
(PCR) assay was designed using two primer pairs: one to amplify the
third exon
and flanking sequences of the α2 gene and the other to amplify a 78-bp
fragment
representing the Southeast Asian (SEA) deletion. Denaturing high
pressure
liquid chromatography (DHPLC) was used to analyze the duplex PCR
products at
63.8°C. The study group included 110 samples (80 α-thalassemia
samples with
various genotypes and 30 normal DNA samples). Results : The PCR products of the sample with
known CS/αα, QS/αα and WS/αα DNA showed significantly different
profiles,
suggesting that DHPLC analysis can directly detect potential mutations.
We also
detected the SEA deletion, αα or αTα alleles, and -α3.7 and -α4.2
deletions.
These results were in accordance with those from multiplex PCR,
real-time
PCR/dissociation curve analysis, reverse dot-blot (RDB) analysis and
DNA
sequencing. Conclusion : A duplex PCR assay followed by DHPLC analysis can
fully diagnose
α-thalassemia. This methodology is simple, rapid and accurate,
semi-automatic
and high-throughput and is thus suitable for large-scale screening. P111 Two
novel approaches used to detect single point mutation causing resistance We have
developed rapid assays for
detection of H274Y in both H1N1 and H5N1 viruses using two novel probe
technologies. For the detection of H274Y in H1N1 we have used
HydrolEasy®
probes. These probes have an increased signal-to-noise ratio and can be
designed to have a specific Tm and thereby be designed to have a better
sensitivity and specificity than equivalent TaqMan® assays. We have
also
introduced a novel end-point detection of H274Y in H5N1 based on the
novel
EasyBeacon™ technology. The end-point detection makes it possible to
use standard
PCR seetings and equipment (non Real-Time) and make a fast (less than
ten
minuttes) end-point reading on a Real-Time instrument. In case of a
pandemic
outbreak is it possible to use this end-point assay for high througput
screnning. In contrary to Molecular Beacons, EasyBeacons™ are easy to
design. The
detection of H274Y alters the recommended treatment and it is therefore
important to test for the presence of this mutation following a
positive H1N1
or H5N1. P112 EXPRESSION
OF A2A AND A2B ADENOSINE RECEPTORS IN HUMAN BREAST TUMORS Background:
After identification of
the expression profile, signal transduction, molecular function and
cell growth
modulation of adenosine receptor subtypes in the human breast cancer
cell
lines, we decided to investigate the possible roles of adenosine
receptors in
the human breast tissues. In this study, we used RT-PCR to assess A2A
and A2B
gene expression in normal and tumor breast tissues. Methods: Breast
tumors and
non-neoplastic mammary tissues (n = 15) were collected immediately
after
mastectomy and stored at -80°C until use. All tumors were
histologically
confirmed to be breast cancer. Total RNA was extracted and reverse
transcribed
to cDNA. PCR primers were synthesized from human adenosine receptor
cDNA
sequences. PCR was performed under optimized condition for each
receptor
subtype. Amplification of beta-actin mRNA served as control for RT-PCR.
The PCR
products were separated on 1.5% agarose gels. Results: To elucidate the
expression of A2A and A2B mRNA in breast carcinoma and normal tissues,
we compared
the level of A2A and A2B mRNA expression by RT-PCR analysis. All breast
tumor
tissue specimens (n = 11) expressed A2A and A2B adenosine receptor
transcripts.
In contrast, only one of the four breast specimens from patients
without
carcinoma expressed no A2A mRNA. Moreover, there were no observable
differences
between normal and tumor tissues, when normalized against that of
beta-actin. Conclusion:
In conclusion, these results indicate for the first time, to our
knowledge, the
expression profile of A2A and A2B adenosine receptors in the human
breast
carcinoma. However, further studies based on the Real-time quantitative
RT-PCR
are needed to identify gene expression levels. P113 Detection
of allergens in spiked pasta by Real-time PCR 1Hochschule
Albstadt-Sigmaringen, Biomedical Engineering; 2Chemisches
und
Veterinäruntersuchungsamt Sigmaringen; panter@hs-albsig.de Food
allergies nowadays represent an
important health problem. Undeclared allergens as contaminants in food
products
pose a great risk for sensitized persons. To ensure compliance with
food
labelling and protection of consumers reliable detection and
quantification
methods for food allergens are required. Yet, the detection of
allergens in
food products can be very challenging, due to the fact that they are
often
present only in trace amounts or are masked by the food matrix.
DNA-based
methods are increasingly used for the detection of foreign food
constituents.
The methods are specific and provide sensitive tools for the detection
of
specific allergenic components in food. Since reference materials (RM)
are
scarcely available for food allergens so far, we took an approach to
detect
five allergens (soy, mustard, celery, lupine, sesame) in spiked pasta
using
single-plex Real-time PCR. Blank pasta was made from durum wheat
semolina and
tested for the presence of the five selected allergens. Then spiked
pasta was
prepared with a starting concentration of 200 ppm for each allergen
respectively. Afterwards dilutions were made using spiked pasta
material and
blank pasta material resulting in concentrations of allergens in spiked
pasta
material of 200, 50, 20, 10, 5, 1 ppm respectively. DNA extraction was
performed using either CTAB or a commercial DNA extraction kit.
Real-time PCR
was performed on Applied Biosystems 7700 or Corbett Rotorgene using
commercial
test kits or published methods. Correlation between Ct value and spiked
amount
of allergen was analyzed for each selected allergen. We showed that
correlations are excellent considering the fact that DNA had to be
extracted
from a food matrix. Limit of detection is about 10 ppm for the selected
allergens in this food matrix. Currently we´re analyzing spiked
spices as
another and more difficult food matrix. P114 Alternative
splicing variants of carbonic anhydese IX in human non-small cell lung
cancer 1Clincal
Biochemistry Unit, Dept. of Clinical Physiopathology, University of
Florence,
Italy; 2Dept. of thoracic Surgery, Azienda Ospedaliera
careggi,
Florence, Italy; 3Institute of Virology, Slovack Academy of
Science,
Bratislava, Slovak Republic; p.pinzani@dfc.unifi.it
In human
cancer, carbonic anhydrase
IX (CAIX) is involved in regulation of intracellular and extracellular
pH under
hypoxic conditions, and also influences regulation of cell
proliferation and
tumor progression. CAIX was previously indicated as an independent
prognostic
marker in non-small cell lung carcinoma (NSCLC). Recently a CAIX
alternative
splicing isoform was detected in cancer cells independently from
hypoxia level.
This alternative splicing variant (AS) derived from a transcript
lacking exon
8-9, generating a truncated protein lacking the transmembrane region,
the
intracellular tail and the C-terminal of the catalytic domain, that
competes
with the full-length isoform (FL) in regulation of the extracellular
pH, mainly
in a mild hypoxic status. In the present study we measured the mRNA
expression
of FL, and AS CAIX isoforms in 101 NSCLC and paired not affected
tissues. We
observed that the two isoforms were coexpressed in all NSCLC and normal
tissues, but AS mRNA was prevalent in normal tissues (66±3%),
while the FL
isoform was higher in NSCLC (58±2%, p=0.001). FL mRNA, and not
AS, was
statistically increased in NSCLC (p=0.01) and showed a statistical
association
with lymphnode involvement (p=0.009) and tumor stage (p=0.04). Global
analysis
of cancer/related death showed that high levels of FL mRNA were
predictive of
unfavourable outcome (p<0.0001) and shorter desease-free survival
(p<0.0001). In NSCLC, multivariate analysis indicated that FL is an
independent prognostic factor for overall survival, and higher levels
of FL
mRNA sensibly increase hazard ratio (approximately six fold). In
conclusion,
our results seems to indicate that FL CAIX is the most accurate
surrogate of
hypoxic stress and represents the only variant with a prognostic role
in NSCLC.
These data indicate the importance of a separate measurement of the two
isoforms in cancer and the need of an accurate re-evaluation of the
most
studies on the clinical role of CAIX in cancer diagnosis. P115 High-
resolution melting analysis for rapid detection of KRAS, BRAF, and
PIK3CA gene
mutations in colorectal cancer 1Clinical
Biochemistry Unit, dept. of Clinical Physiopathiology, University of
Florence,
Florence, Italy; 2Medical Genetics Unit, dept. of Clinical
Physiopathology,
University of Florence, Florence, Italy; 3Dept. of General
Surgery,
University of Florence, Florence, Italy; 4Surgery Unit,
Department
of Clinical Physiopathology, University of Florence, Florence, Italy; 5Dept.
of Preclinical and Clinical Pharmacology, University of Florence,
Florence,
Italy; p.pinzani@dfc.unifi.it
High
Resolution Melting Analysis
(HRMA) provides a new valid approach to efficiently detect DNA genetic
and
somatic mutations. In this study HRMA was used for the screening of 116
colorectal cancers (CRCs) to detect hot-spot mutations on the PIK3CA
gene, KRAS
and BRAF oncogenes. In particular for PIK3CA, mutational hot spots on
exon 9
and 20 were analysed, for BRAF on exon 15 and for KRAS on exon 1.
Direct
sequencing was used to confirm and characterized HRMA results. HRMA
revealed
abnormal melting profiles in 65 CRCs (56.0%), 16 of them harbouring
mutation in
two different genes, simultaneously. The frequency of mutations was
17.2% for
PIK3CA (11.2% in exon 9 and 6.0% in exon 20), 43.1% for KRAS exon 1,
and 9.5%
in exon 15 of the BRAF gene. We found a significant association between
PIK3CA
and KRAS mutations (p=0.008), whereas KRAS and BRAF mutations were
mutually
exclusive (p=0.001). This report describes a novel approach for the
detection
of PIK3CA somatic mutation by HRMA. P116 1Biotools
B&M Labs., Sepsis is a
syndrome commonly called
"blood stream infection" defined by the presence of bacteria
(bacteremia) or other infectious organisms and/or their toxins in the
blood
(septicemia). Sepsis is commonly associated with clinical symptoms of
systemic
illness characterized by a generalized inflammatory response due to the
above
mentioned bloodstream infection, and is one of leading killers in
general
intensive care unit population. For the treatment of septic patients,
it is
important to perform a rapid and accurate identification of the
causative
microorganisms of the syndrome, in order to use the appropriate
medicine.
Growth in liquid media is the conventional method for detecting
microorganisms
associated with this syndrome. However, the blood culture method
requires
several days to detect and identify the bacteria and to run a later
test for
susceptibility to antibiotics. Therefore, in the absence of an
appropriate
diagnostic test, the sepsis-treatment involves broad-spectrum
antibiotics in
advance of the test, which are seriously increasing resistance to
antibiotics
as well as the costs related to its treatment. In the last
years, the development
of qPCR has provided a fast and reliable method for detecting the
presence of
specific taxa in different environments. The aim of this project was to
design
and develop a new sensitive method for the diagnostic of sepsis by qPCR
assay
from potentially positive blood cultures. In order to achieve this
goal, we
have used LionProbes®
, owned by Biotools B&M Labs, We have
adapted the qPCR assay to a
gelified 96-well plate format to increase the throughput and the use of
the
gelification technology in order to stabilize the reaction mixture. Gelification® technology developed by BIOTOOLS
B&M Labs, Here we
present the development and
application of a multi-diagnostic qPCR assay that is less time
consuming and
cost-effective, providing rapid results that allow the application of a
fast
and effective medical treatment, reducing the patient’s exposure to
unnecessary
or ineffective antibiotics. P117 Clostridium
botulinum is the
etiologic agent of botulism, a disease marked by flaccid paralysis that
can
progress to asphyxiation and death. This species of bacteria produces
the most
potent toxins known with an LD50 in primates of 1-10 ng kg-1 of body
weight.
Because of their potency, these toxins have the potential to be used as
biological weapons. Therefore, C. botulinum has been classified as a
select
agent by the United States Centers for Disease Control and is
considered to be
equally dangerous by other governments. There are four related but
antigenically distinct botulinum toxins that cause disease in humans
(A, B, E,
and F) and these can enter the body via three different routes:
inhalation,
ingestion, and absorption from wound infections. Ingestion of C.
botulinum
spores by infants has been associated with sudden infant death
syndrome. The
mouse bioassay is the current gold standard by which toxin type is
confirmed.
However, this method is expensive, slow, and very labor intensive,
taking up to
four days to complete. In addition, this assay carries ethical concerns
due to
the need to sacrifice mice. Commercial biochemical tests have failed in
identifying various toxin-producing strains of C. botulinum. PCR-based
assays
have been used extensively for the detection of botulinum
toxin-producing
bacteria in food, animals, and fecal samples, and recently, to help
diagnose
disease in humans. Most of these are traditional PCR methods, though
assays have
been published in recent years that use real-time PCR to target one or
more
botulinum toxin genes. However, no assay has been published that
involves
real-time PCR detection of the four human disease-causing toxin genes
A, B, E,
and F in a single-tube, multiplex reaction. This report describes the
development of a real-time PCR single-tube assay that uniquely
identifies these
four botulinum toxin types responsible for human disease. A total of 83
C.
botulinum isolates were evaluated in this study, as well as numerous
near-neighbors and other bacterial species. Included were isolates
which had
genes for each of the toxins A, B, E, and F with some natural isolates
containing genes for more than one toxin. C. botulinum isolates
producing the
toxins C and D, which do not cause disease in humans, were also
included as
controls. Results showed that this quadraplexed assay was capable of
detecting
any of the four toxin genes in a given sample at a sensitivity of about
100-200
pg of genomic DNA. Furthermore, it was able to detect the presence of
two,
three, or all four toxin genes in a given sample, indicating the lack
of
type-to-type interference. The test was also functional in the presence
of
extraneous organic matter commonly found in various environmental
samples. This
assay could prove to be a useful tool in the rapid identification of a
specific
type of disease, or the potential toxic threat of a substance to human
health. P118 Alteration
of the CEACAM1-Splice Variant Expression in Bladder Carcinoma 1University
Hostpital Essen, Germany, Department of Anatomy; 2University
Hostpita Essenl, Germany, Department of Urology; 3University
Hospital Grosshadern, Munich, Germany Department of Urology; Inka.Scheffrahn@uk-essen.de
Objective:
Diagnostic and prognostic
biomarkers in bladder carcinoma, which assess early non-invasive
tumours in
their clinical course, help tremendously to decide for the optimal
treatment of
the patient. Carcinoembryonic Antigen-related Cell Adhesion Molecule 1
(CEACAM1) is known to be down-regulated in epithelial cells of various
tumours
and acts as a proangiogenetic factor which could be involved in the
tumour
transition from a non-invasive to an invasive phenotype. The aim of the
present
study was to characterize the four major splice variants of CEACAM1 in
the
progression of bladder cancer and to analyze their prognostic potential
as
tumour markers. Patients and
Methods: 95 cases of
bladder carcinoma patients (30 pTa, 15 pT1, 11 pT2, 29 pT3, 10 pT4) and
four
non-cancer patients were chosen. For each CEACAM1 splice variant a
specific
quantitative RT-PCR was designed and performed. The expression data
were
analyzed using Mann-Whitney-rank sum. Results: It
could be demonstrated
for the first time, that CEACAM1-3L is the predominant splice variant
in normal
tissue. In comparison, all samples of bladder cancer tissue consisted
of
significantly less CEACAM1-3L (p<0,001) and CEACAM1-3S (p=0,006).
Additionally, CEACAM1-4S (p=0,005) and CEACAM1-3S (p=0,013) expression
was
significantly higher in pT1 than pTa. The overall amount of CEACAM1-S
and
CEACAM1-L in normal tissue was the same (ratio S/ L=1,67), while in all
bladder
carcinoma there was significantly more S (ratio S/L >7). Conclusion:
These data suggest, that
the expression of CEACAM1 splice variants change during transition from
normal
transitional epithelium to tumour and probably also from non-invasive
to
invasive and vascularised tumour type. With these properties, the
determination
of CEACAM1 splice variants seems to be a potential marker for bladder
carcinoma. Since CEACAM1 is involved in various other tumours, such as
colon
carcinoma, leukaemia or prostate cancer, it will be valuable to expand
the
characterization of the CEACAM1 splice variants to other normal and
pathological tissues. P119 Dept.
Obstetrics & Gynecology, The
identification of reproductive
toxicants and their mechanisms of action is a major scientific
challenge during
safety assessment of chemicals and the process of drug prioritization.
Our work
is part of the integrated EU-project ReProTect developing new in
vitro tests
required under the new European chemicals legislation (REACH). We
investigate
effects of chemicals interfering with the receptivity of human
endometrium for
embryo implantation. As highly sensitive method qRT-PCR is applied to
detect
chemical effects on mRNA level. As models the human endometrial
Ishikawa cell
line and human endometrial explants cultures are investigated. In order to
identify predictive toxicological
endpoints among a broad spectrum of possible target genes, assays from
the
Universal Probe Library (Roche) were used on a LightCycler 480
instrument. Data
analysis was performed by calibrator-normalized relative quantification
with
efficiency correction. Reference genes were selected by a systematic,
software-based approach (NormFinder, geNORM). Standard curves for low
and
medium expressed genes were established by addition of standard DNA
generated
by conventional PCR to the sample matrix. The
successful operation of the
calibrator-normalized relative quantification method was demonstrated
by
studying mRNA levels of progesterone (PR) and estrogen receptors (ER),
leukaemia inhibitory factor (LIF) and cyclooxygenase-2 (COX-2) before
and after
endometrial explants culture. mRNA levels of PR and ERα were reduced
and those
of LIF and COX-2 increased after 6 hrs of culture. Quantitative
dose-response-relationships were established for the up-regulation of
progesterone receptor mRNA by various estrogenic compounds in the human
endometrial Ishikawa cell line. Specific time courses were elaborated
for the
expression of selected target genes under the influence of various test
substances. Whereas for progesterone and estrogen receptors a
continuous
increase of mRNA levels was found, for LIF a peak mRNA expression after
1-2
hours after addition of progestagenic test compounds was observed. In summary,
qPCR has been
demonstrated to be a sensitive and rapid method for describing
dose-response-relations and time courses of effects of environmental
compounds
on mRNA levels of pre-selected target genes. This project
is funded by the
European Commission (ReProTect, Project no.: LSHB-CT-2004-503257). P120 PROSTATE
CANCER SPECIFIC mRNA/miRNA DETECTION IN CELL-FREE PATIENT URINE BY REAL
TIME
RT-qPCR 1Institute
of Clinical Pathology, LK Weinviertel Mistelbach/Gänserndorf,
Austria; 2University
of Applied Sciences Wr. Neustadt, Department of Biomedical Analytics,
Austria; 3Department
of Urology, LK Krems, Austria; 4Department of Pathology and
Laboratory Medicine, Drexel University, Philadelphia, USA; alfred.schoeller@mistelbach.lknoe.at
Objectives :
Human patient urine contains a cell-bound and a cell-free RNA (ufRNA)
component. Proliferating urogenital tumors (kidney, bladder, prostate)
shed RNA
molecules (mRNA, miRNA) into urine by various physiological mechanisms:
apoptosis, necrosis and cell lysis. We postulate that the prostate
tumor
specific ufRNA fraction could mimic the aberrant cancer RNA expression
signature of the malignant tissue. To investigate the hypothesis we
surveyed selected
biomarker RNAs in the cell free urine supernantant of cancer patients
and a
non-diseased control group using SYBR Green I real time qPCR together
with
validated reference genes. Methods :
Cell-free urine was prepared from consenting young males (aged below 35
years
of age) and patients undergoing routine surgery for benign prostatic
hyperplasia (BPH) and prostate cancer by low speed centrifugation.
Total RNA
extraction was performed with silica filter based protocols (RNeasy
Mini kit,
Qiagen; ZR Viral RNA Extraction Kit, ZymoResearch) and by a
phenol/chloroform
extraction method (peqGold RNA Pure FL). RNA content was quantified
with a
Quant-iT RNA assay kit using a Qubit fluorometer (Invitrogen). cDNAs
were
prepared with a Transcriptor First Strand cDNA kit (Roche Diagnostics)
or a
miRNeasy Mini Kit (Qiagen) and real time PCR experiments were performed
on a
LightCycler® 480 instrument using SYBR Green I for amplicon
detection. The
three best reference genes validated by a statistical geNORM procedure
(GenEx
Professional Software, MultiD) were used for normalizations (geNORM
house-keeping gene selection kit, PrimerDesign). Results : A
geNORM analysis of 9 reference genes with four different urine types
(normal
urine collected from young males aged below 35 years, post-DRE BPH,
pre-DRE and
post-DRE cancer) revealed the overall best fitted gene pair for
relative
quantifications (RPLPO, GAPDH; M = 0.696). From the investigated
markers the
qualitatively most abundant RNAs were PSA, RPS2 and hepsin: normal
(PSA: 2/9;
RPS2: 7/9; hepsin: 1/9), pre-DRE BPH (PSA: 4/5; RPS2: 5/5; hepsin:
3/5),
post-DRE BPH (PSA: 8/9; RPS2: 9/9; hepsin: 4/9), pre-DRE cancer (PSA:
5/16;
RPS2: 15/16; hepsin:4/16) and post-DRE cancer urine (PSA: 14/16; RPS2:
16/16;
hepsin: 7/16). A qPCR real time statistical analysis of the expression
of PSA,
RPS2, five novel patented marker genes (A to E) and six selected miRNAs
(miR-202_2, miR-210_1, miR-320_3, miR-373*_1, miR-498_1, miR-503_1) did
not
significantly differentiate BPH from cancer urine in a small patient
group. Conclusions :
Urine cell-free RNA is a significant diagnostic resource for prostate
cancer
biomarker analysis. We report two novel findings: prostate-derived
ufRNA is
enriched in post-DRE patient urine and the miRNA component represents a
potential novel resource for a diagnostic assay development. (supported
by OENB
P11491). P121 1Blood
transfusion center of Rhesus D
(RhD) blood group
incompatibility between RhD-negative mother and RhD-positive fetus can
occasionally result in maternal alloimmunization where the resultant
anti-D can
cross the placenta and attack the fetal red cells, which in worse case
scenarios can cause fetal anemia and ultimately death. Modern
non-invasive prenatal
diagnostic have been developed using free fetal DNA circulating in the
maternal
blood of pregnant women to determine fetal RHD genotype. The
isolation
and detection of free-fetal DNA is critical because of low yield of
isolated
free fetal-DNA. In our study for isolation of free-DNA the automatic
isolation
by EZ1 instrument (Qiagen) using QIAamp Virus kit (Qiagen) was used. A
new
preanalytic step was included for improvement in test robustness for
reliable
results in early pregnancy. The isolated free-DNA was preamplified with
PreAmp
Master Mix (Applied Biosystems). The real-time PCR TaqMan assays for
exon 7 of RHD gene and/or SRY gene and albumin gene
were
used for
non-preamplified DNA and preamplified DNA. In our study the comparison
between
the different approaches was made. P122 Expression
of IBSP, COL15A1 and WISP-1 in osteoporotic bone tissue 1University
of Studies,
based on the genetics of
osteoporosis, play an important role in clinical practice.
Osteoporosis, a
major public health problem, is a systemic disease of the skeleton,
characterized by low bone mineral density (BMD) and alterations in bone
microarchitecture with a low-energy fracture in the area of hip, wrist
and
spine as a main clinical end-point. Testing for genetic markers in
early youth
could help determine the risk of developing osteoporotic fractures in
later
life. Genes that regulate BMD and bone fragility are potential targets
for the
synthesis of new drugs, which could be used for the treatment of
osteoporosis.
Furthermore, they could be used also as new diagnostic and prognostic
markers
for osteoporosis. A preliminary
study using DNA
microarrays, discovered several genes, which were expressed differenty
in
osteoblasts from osteoporotic as opposed to osteoblasts from
non-osteoporotic
bone tissue. The purpose of our work was to measure the expression of
three
differently expressed genes (IBSP, COL15A1 and WISP-1) in the bone
tissue,
attempting to confirm the involvement of the genes in the pathogenesis
of
osteoporosis. 71 patients
were involved in the
study. Spongious bone tissue was sampled in the proximal femur. BMD was
measured at the total hip, femoral neck and lumbal spine, using
dual-energy
x-ray absorptiometry. qPCR was used to measure the expression of genes
of
interest. PPIA and RPLP were experimentaly choosen as reference genes.
SYBR
Green was used for product detection, with the exception of WISP-1,
where
TaqMan probes were employed. Using standard curves for each gene, we
determined
the relative concentrations of mRNA of each gene in the samples. We
then
normalized the expression of each gene from each individual sample with
the
reference genes. Finally, we statistically evaluated the clinical
significance
of IBSP, COL15A1 and WISP-1 gene expression using non-parametric
statistics. IBSP was
3.2-fold up-regulated in
osteoporotic tissue (p=0.036) when BMD of the total hip was considered.
When
the BMD of the femoral neck and lumbal spine were considered, the
up-regulation
was 2.4-fold (p=0.096) and 1.3-fold (p=0.802), respectively. WISP-1 was
up-regulated only when hip BMDs were considered (4.6 – 3.2-fold
up-regulation),
however it did not reach statistical significance. No difference was
observed
in the expression of COL15A1. Our study
represents a further step
in researching the pathogenesis of osteoporosis, as well as a
contribution to
the development of new solutions for diagnosing and treating the
illnesses of
bone tissue. P123 RAPID
DETECTION OF HUMAN INFLUENZA VIRUSES IN ONE STEP RT-qPCR PORTABLE
MICRODEVICE 1GAIKER, In this work
we have developed a
portable microfluidic device for a specific, rapid and early detection
of human
influenza viruses in clinical samples (nasopharyngeal and throat
swabs). The
microdevice is able to generate cDNA and qPCR amplification of
influenza
molecular markers in one step RT-qPCR taking into account the lab on a
chip
concept. The biochemical reaction is carried out in a l SU-8
microchamber, up
to 40 minutes.msingle
3 Influenza
viruses adapted to human
have caused significant pandemic or epidemic waves. In addition, these
viruses
cause several thousands of deaths each year in The
microfluidic device consists of
a chip (1), with a single microchamber of 3 mm wide and 5.4 mm long for
a 3 μl
RT-qPCR reaction volume. RT-qPCR mastermix containing RNA isolated from
clinical samples is pipetted into the chamber, a chip with electrodes.
The chip
is then introduced into the capsule that is connected to a small device
able to
generate the thermocycling steps. The emitted fluorescence signal is
captured
in real time by a photomultiplier tube through the microchip cover and
fluorescence signal is analysed with a data acquisition unit. The one
step RT-
real time PCR method shows a high specificity and accuracy. The end
point DNA
concentration after RT-qPCR of assays carried out on chip was very
similar to
experiments performed on conventional thermocycler. These preliminary
assays
have been done using universal primers for the amplification of the
matrix gene
of IVA. Next assays will include primers for several sub-types and an
internal
amplification control. This work proves our developed portable
microdevice is
able to carry out the influenza detection by RT-qPCR. In a next future
we will
include a RNA isolation step from clinical samples, in order to provide
a more
integrated lab on a chip microdevice. The research
leading to these
results has received funding from the European Community's Seventh Framework
Programme ([FP7/2007-2013]
under grant agreement n° 201914 Portfastflu. We would like
to thank the
Microbiology Department of Hospital Donostia for supplying clinical
samples. (1)
Aguirregabiria et al.,
Proceedings of uTAS 07, 584-586 (2007) P124 Expression
levels of type III and VI mRNA transcripts from TMPRSS2-ERG gene fusion
in
cancerous and benign prostate tissue by qRT-PCR 1Department
of Biotechnology, University of Turku, Turku, Finland; 2Department
of Pathology, University of Turku, Turku, Finland; 3Department
of
Surgery, University of Turku, Turku, Finland; ripava@utu.fi OBJECTIVES -
Novel prostate cancer
markers are needed for improved diagnosis and prognosis of this
heterogeneous
disease. Transcripts from a gene fusion between TMPRSS2 and ERG
have
lately been discovered in subgroups of prostate cancers. Our aim was to
study
the clinical applicability of the most frequently expressed transcript
(type
III) and the transcript suggested to be associated with aggressive
prostate
cancer (type VI) by determining their expression levels in benign and
malignant
human prostate tissue samples obtained immediately after radical
prostatectomy.
The expression levels were also compared to KLK3 mRNA counts. METHODOLOGY -
Quantitative real-time
reverse-transcription PCR (qRT-PCR) assays were developed for TMPRSS2-ERG
transcripts
types III and VI. Assays were based on a generic closed-tube protocol
developed
at our department. The real-time detection in PCR is based on
time-resolved
fluorometry by utilizing lanthanide chelate-labeled target specific
probes and
separate quencher probes. In addition to using external standard
curves,
artificial RNA molecules are added to samples to serve as internal
standards. A
sample panel consisting of GITC-stabilized radical prostatectomy tissue
samples
from 88 prostate cancer patients was analysed. Two samples were
collected from
each prostate aiming at the cancerous site and a benign control sample.
Microscopic examination of the adjacent tissues was performed to
confirm the pathology
of the samples. Also, expression level of KLK3 mRNA was
determined with
a similarly constructed assay from the same samples. RESULTS -
Detection limits for the TMPRSS2-ERG type III, TMPRSS2-ERG
type VI and KLK3 mRNA
assays were 5, 20
and 2.5 mRNA copies per µl of cDNA template, respectively. Using
binary
classifications, fusion transcript type III was found in 42 % of the
benign
samples and in 56 % of the cancerous samples (p=0.06) and type VI was
found in
30 % in the benign group and in 51 % of the cancerous samples
(p=0.007). The
fusion transcript concentrations per µg RNA of the positive
cancerous and
benign samples did not differ significantly from each other (type III,
p=0.10
and type VI, p=0.45). KLK3 mRNA expression was not found to be
changed
from benign to malignant tissue. CONCLUSIONS -
This is a first report
of a truly quantitative assay for the determination of TMPRSS2-ERG type
III and type VI fusion transcripts in cancerous and benign tissue
samples
obtained early after radical prostatectomy. The frequency of prostate
cancer
samples positive for the TMPRSS2-ERG fusion transcripts is
similar to
previously reported results. The high positivity and expression levels
of the
fusion transcripts in the benign tissue samples was unexpected, and may
be
explained by lack of true non-cancerous homogeneity and/or a field
effect from
the cancer site. Further studies with samples from non-cancer patients
are
needed to resolve this question. P125 Introducing
a new biomarker for schizophrenia Islamic Azad
University of
Tonekabon, Iran (Islamic Republic of); reza_sonboli@yahoo.com Schizophrenia,
commonly developed in
adolescents and young adults, is one of most common mental disorder,
but the
phatophysiology and etiology of schizophrenia is still obscure.
Numerous
studies on dopamine and schizophrenia have suggested that change in the
dopamine system is related to schizophrenia, but there is little direct
evidence
for dopamine hypothesis in schizophrenia. Recent
progress in molecular
biology, and imaging technique has enable new insight for schizophrenia
research. Changes in the dopamine system are influence not only by
dopamine
itself, but also by dopamine receptors. Recent
progress in molecular biology
reveals the existence of mRNA of D3 and D4 dopamine receptor in
peripheral
lymphocyte. However, the clinical significance of these finding and
whether or
not these receptor reflect central dopamine receptors remains uncertain. The purpose
of this study were to
examine if the mRNA of the peripheral dopamine receptor is changed in
schizophrenia patients. 50 Naive
patients were enrolled.
After extracting RNA from white blood cells, cDNA is synthesized. After doing
the quantitative
Real-time PCR, expression of D3 receptors in healthy persons and
patients were
compared. Results of
this examinations reveals
that expression of D3 receptor is increased in compared with controls
sample. P126 Differential
Expression of hypothetical genes in a biochemical morphotype of the
marine
sponge Discodermia dissoluta 1Nova
Southeastern University, Dania Beach FL, USA; 2Florida
Atlantic
University at Harbor Branch Oceanographic Institution, Fort Pierce, FL,
USA; 3Center
for Bioinformatics and Computational Biology, University of Maryland,
College
Park, MD, USA; 4SymBio Corporation, Menlo Park, CA, USA; waikel@nova.edu The marine
sponge Discodermia
dissoluta has been shown to produce a variety of secondary
metabolites,
some with promising antitumor properties such as discodermolide.
Suppression
subtractive hybridization (SSH) experiments with two D.dissoluta biochemical
morphotypes have yielded a large expressed sequence tag (EST) dataset
of 1,536
transcripts including eukaryotic, prokaryotic, and viral genes.
Preliminary
analysis of the resulting transcripts indicated a profile of 880
eukaryotic,
325 prokaryotic, 21 viral, and 44 unknown ESTs. Of these, 538 represent
hypothetical genes, some of which are the focus of this study. We have
analyzed differential gene
expression using quantitative polymerase chain reaction (qPCR) with
SYBR Green
chemistry to screen and characterize genes, some of which may be
related to
novel metabolism and unknown functions related to symbiosis within the
complex
sponge-microbial community. Preliminary data indicate differential
expression
of selected hypothetical genes in the secondary metabolite positive
morphotype.
Bioinformatics analysis of these transcripts also provides insight into
their
potential function and ecological roles within the complex sponge
microcosm. This
information will in turn allow
researchers to screen for novel drug candidates by probing for specific
genes
involved in secondary metabolite production. Screening for specific
genes
requires substantially less material than traditional analytical
chemistry
methods which require a large amount of biomass. Additionally,
elucidation of
the genes potentially driving the production of bioactive substances
will aid
in their synthesis in the laboratory. This will eliminate the need for
large
scale harvesting of organisms, thereby conserving the natural
environment. P127 Highly
sensitive qPCR for the JAK2-V617F mutation to guide adoptive
immunotherapy in
patients with myelofibrosis who received allogeneic stem cell
transplantation 1University
Medical Centre Hamburg-Eppendorf, Germany; 2Medical School
Hannover,
Germany; 3University Hospital Cologne, Germany; badbaran@uke.de During the
last few years allogeneic
stem cell transplantation (allo-SCT) after dose-reduced conditioning
has been
established as a promising treatment option for myelofibrosis. However,
relapses of the malignant disease after transplantation remain a
significant
problem. Single-case reports on successful donor lymphocyte infusions
(DLI) for
relapse treatment after allo-SCT have provided evidence for a
graft-versus-myelofibrosis effect. In this study we investigated the
potential of
our recently developed highly sensitive JAK2-V617F-specific real-time
PCR to
guide adoptive immunotherapy in myelofibrosis patients after allo-SCT.
Therefore, the clinical impact of escalating DLI was compared in 17
patients
with myelofibrosis who had either a clinical relapse (salvage-DLI, n=9)
or a
molecular relapse / molecular residual disease (pre-emptive DLI, n=8)
after
dose-reduced allo-SCT. Thirty-five DLI from related (n=5) or unrelated
donors
(n=12) were given with a median dose of 1x106 for the first and 5 x 106
CD3+cells/kg for the second DLI. The complete molecular response rate
was 68 %,
and significantly higher for patients who received DLI for molecular
than for
clinical relapse (100 % vs. 44 %) (p=0.04). In comparison to molecular
relapse,
clinical relapse required more donor-lymphocyte infusions (two vs. one)
and was
associated with a higher incidence of acute GvHD (22 % vs. 0 %).
Notably, two
of the four patients who achieved complete molecular remission in the
salvage
group had developed an acute GvHD (grade III), whereas no GvHD was
documented
in non-responders. These observations confirm earlier data on a strong
donor T
cell-mediated graft-versus-myelofibrosis effect. In conclusion, the
molecular
monitoring of minimal residual disease using a highly sensitive qPCR
for the
JAK2-V617F-mutation represents an efficient mean to guide adoptive
immunotherapy in myelofibrosis patient. This approach increases
clinical
efficacy of donor-lymphocyte infusions while decreasing their toxicity.
We therefore
suggest that qPCR-triggered adoptive immunotherapy should be
implemented in
further clinical trials. P128 NICOTINAMIDE
N-METHYLTRANSFERASE: A POTENTIAL PROGNOSTIC MARKER FOR ORAL SQUAMOUS
CELL
CARCINOMA. 1Università
Politecnica delle Oral squamous
cell carcinoma (OSCC)
is the most common malignancy of the oral cavity, representing 90 % of
all oral
cancers. Despite advances in diagnosis, surgical techniques, general
patient
care, and local and systemic adjuvant therapies, the mortality rate of
OSCC has
shown little improvement over the last three decades and the overall
five-year
survival of these patients remains less than 50%. The high mortality
from OSCC
is attributed to the presence of cervical lymph node metastasis and the
diagnostic delay seems to be responsible for the poor prognosis of
patients
with OSCC. Therefore, there is an imperative need for sensitive
biomarkers to
improve early detection of this malignancy. In the
present study, we focused on
the expression of genes critical in the drug metabolism process, namely
on
Nicotinamide N-Methyltransferase (NNMT), enzyme belonging to Phase II
Metabolizing Enzymes. To explore the involvement of NNMT in OSCC, we
analysed
the enzyme expression in paired tumour (T) and non-tumour (NT) tissues
obtained
at surgery by semiquantitative RT-PCR, Real-Time PCR, western blot and
immunohistochemical analyses. Compared with
normal mucosa, OSCC
exhibited significantly increased expression of NNMT in 11 of 22 (50 %)
examined patients. Interestingly, NNMT was upregulated in most of the
favourable OSCCs (N 0), while no marked NNMT expression alterations
between
tumour and normal mucosa were detected in most of the unfavourable
OSCCs (N+).
Both, pT and pathological staging showed an inverse correlation with
NNMT mRNA
levels, and a significant negative association of the amount of NNMT
expressed
by tumour tissue compared to the adjacent normal mucosa was found with
metastasis (1). We also
evaluated the effect of
shRNA-mediated inhibition of NNMT on the proliferative potential and
apoptosis
of oral cancer cell line PE/CA-PJ15. The efficiency of gene silencing
was
detected by Real-Time PCR and western blot analysis. The cell
proliferation inhibition
was determined by MTT and soft agar colony formation assays; cell cycle
distribution and apoptosis were examined by flow cytometry. ShRNA
vectors
targeted against NNMT efficiently suppressed gene expression, showing
inhibition rates around 70 %, observed at both the mRNA and protein
levels. The
shRNA-mediated gene silencing of NNMT resulted in a significant rise in
apoptosis rate. The present
data support the
hypothesis that the enzyme plays a role in tumour expansion, and NNMT
expression level measurements would provide a rapid and useful method
of
identifying patients at high risk of lymph node metastases. Therefore,
NNMT may
have potential as a new prognostic marker, and its inhibition could
represent a
possible molecular approach to the treatment of OSCC. 1. Sartini D
et al. (2007) MOL MED
13, 415-421. P129 NEUROTROPHINS
AND RECEPTORS EXPRESSION PROFILING OF PLACENTAS FROM PREGNANCIES
COMPLICATED BY
HELLP SYNDROME AND INTRAUTERINE GROWTH RESTRICTION (IUGR) Università
Politecnica delle OBJECTIVE:
The neurotrophin family
comprises molecules involved in growth, differentiation, survival,
diseases, regeneration
and normal functions of the neuronal system. Particularly,
neurotrophins play
an important role in pre-natal and post-natal brain development due to
their
neuro-protective action. Our aim was
to investigate the
expression pattern and the role of neurotrophins and their receptors in
the
placentas from pregnancies complicated by HELLP (Hemolysis, Elevated
Liver
enzymes and Low Platelet count) syndrome and intrauterine growth
restriction
(IUGR). STUDY DESIGN:
Placentas from normal
term pregnancies (n=15), pregnancies complicated by HELLP syndrome
(n=10) and
intrauterine growth restriction (IUGR) (n=10) were collected. Macroarray
analyses were performed
with GEArray Q Series Human Neurotrophin and Receptors Gene Array
HS-018
(SuperArray Bioscience Corporation, Frederick, MD). The data were
confirmed by
quantitative Real-Time PCR. The Student’s test was used for statistical
analysis. Differences were considered significant at p < 0.05. RESULTS: The
expression of 10
(HELLP) and 6 (IUGR) genes, respectively, was significantly different
in the
pathological group compared to control. Particularly, 3 genes were
up-regulated
and 3 down-regulated in the IUGR group while 6 genes were up-regulated
and 4
down-regulated in the HELLP group. Differential gene expression
measurements
(HELLP versus normal, and IUGR versus normal), performed by real-time
PCR
technique, revealed a significant down-regulation (P<0.05) for STAT3
(signal
transducer and activator of transcription 3) and its isoforms STAT3α
and STAT3β
(fold change values between 1,13 and 2,32). CONCLUSIONS:
A variable capacity of
producing some neurotrophins could be involved in the pathogenic
mechanism
leading to pregnancies complicated by HELLP syndrome and IUGR. Our data seem
to suggest that STAT3α
could play a key role as common denominator for all important factors
involved
in successful pregnancy outcome (e.g. “IL-6 cytokine family”). In addition,
the observed STAT3β
down-regulation underlines the relevance of this isoform in regulating
a
different subset of target genes, which appear play trophic roles in
decidual
development. P130 Direct and Fast DNA Methylation QuantitationLife
Technologies, DNA
methylation is the most
important epigenetic alteration involved in normal cellular processes
as well
as diseases. In cancer research, aberrant DNA methyaltion has been
discovered
as marker for cancer diagnostics and prognosis. Detection and
quantitation of
methylated DNA often starts with bisulfite conversion of non-methylated
cytosines to uridines, leaving methylated cytosines unchanged. The
differences
between methylated and non-methylated sequences can be then analyzed by
various
assay formats. We have
developed a universal
bisulfite conversion protocols to convert cytosines in DNA that are
either
purified or in cells. The whole process, including cell lysis,
bisulfite
reaction, desulfination, and purification, can be completed in two
hours. In
combination of high resolution melting analysis, down to 1% methylated
DNA can
be quantified directly from cell mixtures. Application
of qPCR Assays to the Investigation of New Classes of Antiretroviral
Drugs in
the Treatment of HIV/AIDS McGill
University AIDS Centre, Lady
Davis Institute for Medical Research, Jewish General Hospital,
Montreal,
Canada; aaron.donahue@gmail.com
Background: Highly
active antiretroviral
therapy (HAART) has been very successful in the treatment of HIV-1
infection.
However, due to rapidly emerging drug-resistant virus, new classes of
drugs are
continually required, ideally targeting a different stage of the
retroviral
lifecycle. Drugs that target the HIV-1 integrase, a critical enzyme for
viral
replication, have recently been approved for use. Due to their unique
action
against this stage of viral replication, clinical trials with HIV-1
integrase
inhibitors have shown excellent results. Paradoxically, by preventing
integration, integrase inhibitors result in a build-up of unintegrated
DNA that
is transcriptionally active and can produce early HIV-1 gene products,
with
unknown consequences. Methods and Results:
We have
developed Taqman probe-based qPCR assays to quantify different DNA
forms
present throughout the retroviral lifecycle, including early and late
reverse
transcription products, unintegrated DNA circles, and integrated DNA.
We have
also developed a qRT-PCR assay to detect all of the ~23 mRNA splice
variants of
early HIV-1 gene products, which can be produced from unintegrated DNA.
These
assays have been applied to cell culture experiments involving
treatment of
CD4+ T cells or macrophages with inhibitors of HIV-1 reverse
transcriptase or
integrase. Results will be presented that show the impact of inhibiting
different stages of viral replication, in an aim to dissect the
enhanced
clinical efficacy shown by HIV-1 integrase inhibitors. Additionally,
results
will be presented showing the effect that inhibiting integration with
clinically approved antiretroviral drugs has on the production of early
viral gene
products from unintegrated DNA, to complement other studies by our
group into
their potential effects on both infected and uninfected cells. Discussion: By applying q(RT)-PCR assays to cell
culture experiments that include the treatment of HIV-1 with different
classes
of antiretroviral drugs, we hope to further understand the extremely
promising
results shown by integrase inhibitors for the treatment of HIV/AIDS.
This will
be useful in the development of future antiretroviral drugs, and hopes
to shed
light on strategies aimed at limiting the establishment of the latent
reservoir
of HIV-1, which represents the ultimate barrier to eradication of HIV-1
infection. P132 Development
of a RQ-PCR Assay for quantitative detection of viruses like Simian
Virus 40,
as well as for dental pathogens like Streptococcus mutans 1University
medical center Hamburg-Eppendorf, Clinic and Policlinic for Paediatric
Haematol. & AIM: Simian
virus 40 (SV40)is known
to posess strong oncogenic potential, based on the large T-antigen
(TAG).
Previous studies detected SV40 in a variety of human malignancies, so
in
lymphomas, and osteosarcomas, but with significant discrepancies in its
frequency. Differences in DNA quality and in virus detection methods
hamper a
controlled and standardised analysis. We establish a RQ-PCR based
TaqMan assay
for rapid and highly reproducible detection and quantification of SV40
and used
it for analysing samples from childhood malignancies. Streptococcus
mutans (S.
mutans) has been consistently linked with the formation of dental
caries, one
of the most common infectious diseases afflicting humans. Moreover it
is
associated with non oral infections, so to endocarditis. Various
methods have
been developed to identify oral bacteria, but rare quantitative
analysis are
done up to now. In spite of the importance of S. mutans as cariogenic
dental
pathogen our aim was to establish a RQ-PCR based TaqMan assay for
detection and
quantification of S. mutans in oral specimens. METHODS: DNA was
extracted
either from fresh cells, or dental plaque, using the Quiagen spin
column
method. RQ-PCR primer were selected in case of SV40 for the
retinoblastoma
pocket binding domain of TAG and in case of S. mutans for the
glucosyltransferase
B (gtfB)-gene. The internal TaqMan probes were each labelled with FAM
at the
5`end and TAMRA at the 3`end. RQ-PCR was performed in 10µl
reactions with 500ng
template DNA, 1 U Taq-Polymerase, and annealing temp. of 60oC (SV40) or
58oC
(S. mutans. DNA was serial diluted (10-1 to 10-6) and samples tested
double or
triple. Positive samples were analysed additionally on agarose gels,
and in
part sequenced by using the Abi Prism BigDye terminator cycle
sequencing kit. RESULTS:
For SV40 we analysed tumour-, blood- and bone marrow- samples from a
variety of
malignancies as well as blood from healthy donors with this real-time
quantitative assay. Overall we found SV40 virus in 22% osteosarcomas,
76%
lymphomas, 21% Breast cancer and 1,4% healthy individuals from German
origin.
In samples from Hungarian origin we found in 74% osteosarcomas and 18%
healthy
individuals SV40 positivity. For S. mutans we analysed plaque samples
with this
RQ-PCR assay from a variety of donors, those who consumed chewing gums
with sugar
or with Xylit as a sugar substitute, and compared both groups with
those never
using chewing gum, in order to get quantitative differences in S.mutans
bacteria. CONCLUSIONS: Here we present a RQ-PCR based assay for
standardised
detection and quantification of virus as well as bacteria sequences
which can
also be used for detection and quantification of other viruses or
bacteria. The
use of 500ng template DNA in each reaction and the exact quantification
allows
the sensitive detection of virus sequences down to 4 log as well as for
bacteria down to 6 log. Financially
supported by Kinderkrebszentrum P133 Differential
gene expression analysis of APP isoforms in platelets from patients
with
Alzheimer’s Disease and healthy controls. Università
Politecnica delle Alzheimer
disease (AD) is a chronic
neurodegenerative disorder characterized by a progressive cognitive and
memory
decline. In the past few years, different biochemical parameters in
cerebral
spinal fluid (CSF) or plasma have been investigated. It has been tried
to identify
peripheral markers of AD, focusing mostly on the amyloid precursor
protein
(APP). Different isoforms of APP are generated by alternative splicing.
The
three major isoforms are constituted of 770, 751 and 695 aa residues.
APP 751
and APP 770 contain a Kunitz-type serine protease inhibitor domain
(APP-KPI),
and APP 695 lacks this domain. In this regard, platelets represent an
important
peripheral source of APP. It has been demonstrated that three major APP
isoforms with apparent molecular weight ranging from 106 to 130 kDa are
inserted in the membrane of resting platelets and that both platelets
and
megakaryocytes express three transcripts encoding for the isoforms: APP
770,
APP 751, APP 695. Several studies independently described alterations
in APP metabolism/concentration
in platelets of Alzheimer’s Disease patients when compared to control
subjects
matched for demographic characteristics. These observations define the
frame of
the present study, which aims to investigate the expression level of
different
platelet APP isoforms using Real-Time PCR in patients affected by AD
(n=20) and
in age-matched controls (n= 10). Differential gene expression
measurements (AD
versus control) revealed a significant up-regulation for total APP
(1.49-fold),
for APP-KPI (1.57-fold), for APP 770 (1.37-fold), for APP 751
(1.39-fold) and
for APP 695 (1.33-fold). In the present study we have demonstrated that
patients with AD are characterized by differential levels of
platelet-derived
APP isoforms, thus suggesting that APP could be considered a potential
peripheral marker for diagnosis of Alzheimer disease. P134 Discovering
mechanisms of toxicity and related biomarkers with qPCR Vrije One of the
main aims of the VU
University Amsterdam’s Institute for Environmental Studies (IVM) is to
contribute to solutions in the field of environmental pollution. Its
position
in this field is unique because of the various disciplines that are
combined in
our laboratory: analytical and environmental chemistry; toxicology;
molecular
biology; and ecology. Using an analytical chemical approach, we perform
analysis of low levels of various environmental contaminants, including
classical organic contaminants and emerging contaminants such as
endocrine
disrupting compounds. Our toxicological research focuses on determining
the
biological effects of (mixtures of) chemicals in the environment using
both
novel and classical toxicological approaches. The development of rapid
and
sensitive biomarkers to profile the exposure and effects of chemicals
is an
important pillar of our research. Quantitative RT PCR has proven to be
a
sensitive and rapid method to identify chemicals in the environment
with
specific toxic mechanisms of action. For example, we have developed
qPCR assays
to quantify the expression of genes involved in thyroid hormone
metabolism and
transport, and identified chemicals that alter expression of transport
proteins
and metabolic enzymes. Along these lines, we have shown for the first
time that
chemicals present in the blood of Arctic mammals (polar bears, seals)
have the
potential to induce the expression of thyroid hormone metabolizing
enzymes,
which may play a role in reduced thyroid hormone levels found in wild
populations. Chemical analytical measurements are ongoing to reveal the
identity of the chemicals likely causing this effect and help to
validate the
developed qPCR assays as biomarkers of thyroid hormone disruption. P135 Evaluation
of ARMS PCR when combined with limiting dilution assay for K ras
mutation
detection. Background:
Different techniques
have been used to detect mutant K-ras2 when it is mixed with wildtype
(K-RAS2)
DNA in order to diagnose pancreatic cancer at an early stage. The
Amplification
Refractory Mutation System (ARMS) has the potential to discriminate
alleles and
therefore identify point mutations and various variants of this
approach have
been applied. However, the results have shown poor reproducibility and
in most
cases have not achieved the sensitivity or specificity required for
clinical
use. In this study we have developed a modified ARMS technique by
combining it
with limiting dilution PCR in order to improve sensitivity and
specificity.
Limiting dilution involves diluting mixed populations of DNA and then
diluting
to one or two molecules per individual PCR reaction: This gives the
potential
to obtain individual reactions which have very high concentrations of
low
abundance sequences by carrying out many duplicate reactions. Materials
and
Methods: DNA was obtained from a cell line (PANC-1) which contained a
known
K-Ras mutation (G12D) and from control blood which was believed to have
no
mutant K-Ras (wildtype DNA, G12G). DNA was diluted to a stock
concentration of
10ng/ µl. Mixtures of these DNA species were made (e.g. mix 1:10
having 1 part
mutant to 10 part wildtype DNA). Pure and mixed DNA was serially
diluted and
PCR amplified with primers specific for G12D (aspartate primer set) or
primers
that were not allele specific (control primer set). Amplification was
monitored
in real time using the LightCycler 480 machine and at end point using
gel
electrophoresis. Results: The limit of detection was found to be at a
dilution
0.001 for PANC-1 and 0.01 for control genomic DNA. Reproducibility was
tested
with both control and aspartate primer sets by repeat measures and as
expected
the log of dilution factor of PANC-1 DNA gave a proportional increase
in
crossing point (Cp) in real time PCR (linear coefficient of between 1.8
and 2.4
and R2 of between 0.82 and 0.95). However, when mix 1:10 was diluted
and
assayed there was an inverse relationship between log dilution factor
and Cp
(linear coefficient of between -0.64 and -0.62 and R2 of between 0.40
and
0.41). This means that the greater the dilution (lower actual
concentration)
the higher the apparent concentration appeared. Conclusion:
Quantification
depends on the concentration of wildtype as well as mutant K-Ras2
suggesting
that the wildtype sequence is inhibitory to the mutation specific PCR.
Limiting
dilution will therefore give greater apparent levels of mutant sequence
even
before a jackpot amplification occurs and this must be taken into
account when
analyzing data. A dilution curve may in future replace single point
analysis
allowing more standard quantification of mutant sequence rather than
misamplification of wildtype template.This may allow improved
specificity and
sensitivity of the ARMS assay for the detection of cancer. P136 1Unit
of Medical Statistics and Biometry, Fondazione IRCCS Istituto Nazionale
Tumori,
Milan - Italy; 2Preclinical Chemotherapy and Pharmacology
Unit,
Fondazione IRCCS Istituto Nazionale Tumori, Milan - Italy; paolo.verderio@istitutotumori.mi.it
The
optimization of treatment with
antitumor drugs remains a major effort of preclinical and clinical
research.
The over-expression of ATP binding cassette (ABC) transporters by tumor
cells
has been recognized as a mechanism contributing to the poor response of
tumor
cells to structurally and mechanistically unrelated antitumor drugs in
experimental models. Whole genome approaches have documented the
existence of a
wide family of ABC transporters in human cells. Quantitative analysis
of the
expression of ABC transporters in cell lines characterized with respect
to the
pattern of response to clinically useful antitumor agents may be useful
to
define those genes that can be associated with the multidrug resistant
(MDR)
phenotype. In the present study, we used Taqman Micro Fluidic Cards to
identify
the ABC transporters transcripts that were associated with the
drug-resistant
phenotypes of ovarian carcinoma cell lines developed in vitro after
exposure to
cisplatin (IGROV-1/Pt1) or to camptothecin (IGROV-1CPT/L) and
characterized by
a complex pattern of resistance to multiple agents. The expression of
ABC
transporters in each of the two cell variants was evaluated relatively
to the
parental IGROV-1 cell line in two independent experiments. A
statistical
procedure based on multivariate approaches and re-sampling techniques
was
implemented to process expression values of ABC transporters. According
to our
analysis, a group of seven ABC transporters may be implicated in
conferring
multidrug resistance to the IGROV-1/Pt1 and IGROV-1CPT/L cell variants.
The MDR
gene signature identified in our preclinical model, throughout an
ad-hoc
statistical procedure, may be tested in clinical samples from ovarian
cancer
patients in an attempt to identify genes implicated in clinical
resistance to
chemotherapy. P137 FBP1
is overexpressed in Hepatocellular Carcinoma and regulates the
expression of
cell-cycle and apoptosis-related genes 1Georg-Speyer-Haus,
In a
functional yeast survival
screen, we isolated a cDNA coding for a C-terminal fragment of FUSE
Binding
Protein 1 (FBP1) from a mamma carcinoma cDNA library. FBP1 has
previously been
described as a transcriptional regulator of the proto-oncogene c-myc,
which is
downregulated during cell differentiation. Preliminary experiments
confirmed an
anti-apoptotic effect of FBP1 in mammalian cells. Because FBP1
activates an
important oncogene and at the same time protects cells from apoptosis,
we
analysed expression of FBP1 in tumors by Immunhistochemistry. We found
strong
nuclear staining for FBP1 in 83% (90/109) of the analysed Hepatocelluar
Carcinoma, whereas in normal liver tissue no nuclear staining was
observed. Stable
knockdown of FBP1 in the HCC
cell line Hep3B strongly reduced tumor growth in a xenograft
transplantation
mouse model and we could show decreased proliferation and increased
sensitivity
to apoptosis for these cells in vitro. To identify
the molecular mechanism
responsible for the observed effects following knockdown of FBP1, we
analysed
the mRNA expression of cell cycle- and apoptosis related genes by Real
Time
PCR. Using the Human Cell Cycle Regulation Panel, 96 (Roche), we found
a
significant upregulation of the cell cycle inhibitors p21 and p15 as
well as
downregulation of Cyclin D2. Differential expression of these genes
might cause
the reduction in cell proliferation. Surprisingly, the expression level
of the
published FBP1 target gene c-myc was not significantly changed in these
cells,
indicating that regulation of c-myc expression might be independent of
FBP1 in
liver cells. Expression analysis of apoptosis-related genes using the
Human
Apoptosis Panel, 384 (Roche), revealed upregulation of the
pro-apoptotic genes
Bik/Nbk, Noxa, TNF-α and TRAIL. The observed changes in gene expression
are in
line with the observed behaviour of cells and we are currently
analysing wether
these differentially regulated genes are direct targets of FBP1. Taken
together, our data suggest
FBP1 as a new important factor in the formation of Hepatocellular
Carcinoma and
we assume that the effect of FBP1 is mediated by the (direct or
indirect)
regulation of genes involved in the control of cell proliferation and
apoptosis. P138 Investigation
of Lineage-Specific Chimerism in Post-Stem Cell Transplant Patients 1Department
of Molecular Pathology, Southampton University Hospitals NHS Trust,
Southampton, United Kingdom; 2Department of Haematology,
Southampton
University Hospitals NHS Trust, Southampton, United Kingdom; rhg@soton.ac.uk Objectives The
aim of this study was to investigate the utility of lineage-specific
‘chimerism’ for the improved management of post-stem cell transplant
patients.
After transplant, an assessment of engraftment can be made by
monitoring the
proportions of donor and recipient cells in a sample of whole blood.
Determining the mix of cells, or ‘chimerism’, aids the scheduling of
specific
therapeutic interventions. Here we extended our analysis of whole blood
chimerism to a lineage-specific test, aiming to provide additional
information
about the dynamics of engraftment and facilitate more effective
treatment in
the post-transplant period. Methodology Whole blood
chimerism was measured
by tracking a simplified genetic fingerprint of polymorphic short
tandem
repeats (STR’s) that uniquely defined the donor and recipient. PCR
reactions
using 3 STR markers were set up using a commercially available
forensics kit.
The differently sized fluorescent PCR products were detected and
analysed on a
capillary system genetic analyser. An assessment of the proportions of
donor
and recipient chimerism was calculated from the analyser output using a
simple
algorithm measuring the peak height and area. For the lineage-specific
analysis, leukocyte sub-types were isolated by cell separation using
AutoMACS®
immuno-magnetic separation technology. DNA was isolated from these
purified fractions
and levels of chimerism analysed as for whole blood. Results Validation
tests using control
samples indicated chimerism could be accurately assessed in the
different
leukocyte lineages. Of eight patients studied, two patients displayed
full
donor chimerism in both the whole blood and lineage-specific analysis.
Three
patients with whole blood donor chimerism of <100% showed more
pronounced
fluctuations in lineage-specific donor chimerism. One patient, who
consistently
showed whole blood donor chimerism of 90-95%, displayed a similar level
of
donor chimerism in the myeloid fraction (CD33), but a more significant
decrease
to 55% in the T-cell (CD3) fraction. This patient received a stem cell
transplant after refractory chronic myeloid leukaemia and so this
result could
give useful information as to whether the chimeric state was due to a
recurrence of the original myeloid clone. Reduced T-cell chimerism is
strongly
associated with graft failure and low donor CD3 value could also
provide
evidence as to the success of the engraftment. Conclusions This study
confirmed that
lineage-specific chimerism analysis represents a valuable adjunct to
whole
blood studies. Due to its increased sensitivity it can be used to
reveal mixed
chimerism in specific leukocyte populations that are masked in the
whole blood
analysis. Information about the relative proportions of donor and
recipient
T-cells is important in understanding the dynamics of engraftment and
predicting graft vs leukaemia and graft vs host effects, and so T-cell
chimerism represents a particularly valuable part of the analysis. P139 Lack
Of Mother-To-Newborn Transmission Of Hepatitis C Virus In Iraqi Women -
A
Prospective Study With Hepatitis C Virus RNA Testing 1Dept.of
Community Medicine, AL-Nahrain Faculty of Medicine, Baghdad -Iraq; 2Dept.
of Obstetrics and Gynecology, AL-Nahrain Faculty of Medicine, Baghdad -
Iraq; waqar_abd@yahoo.co.uk
Background:
What has been published
about the risk of mother-to-infant transmission of hepatitis C virus
(HCV),
shows variation according to the population studied and the test used.
Polymerase chain reaction (PCR) was used for the first time in Material
& methods: HCV
antibodies (Abs) were sought with third generation enzyme immunoassay
(EIA-3)
in 3491 pregnant women. A positive reaction was then confirmed by a
third-generation immunoblot assay (LiaTek-III). This last test was
confirmed
positive in 112 serum samples . we followed 26 babies of 25 anti-HCV
positive
mothers at first month of life. Eight of these children could be
followed for
six months postnataly. Result: All
the26 neonates were
positive for HCV Antibodies (with EIA-3 and Lia Tek-III) during the
first month
of life and it completely disappeared within the following six months.
HCV RNA
was consistently negative in 22 sera (14 infants at first months and 8
of
repeated at 6 months later) regardless of the hepatitis C virus
polymerase
chain reaction status of their mothers (9 of whom were positive for HCV
RNA). Cnclusion:
The study showed the
absence of vertical transmission of HCV from pregnant Iraqi women to
their
offspring. P140 Measuring
infarct size using real-time RT PCR 1Lillehammer
University College, Lillehammer, Norway; 2Institute for
Experimental
Medical Research, University of Oslo, Oslo, Norway; 3Department
of
Molecular Biosciences, University of Oslo, Oslo, Norway; 4Cardiothoracic
Surgery, Ullevål University Hospital, Oslo, Norway; 5Surgical
Division, Ullevål University Hospital, Oslo, Norway; 6Deptartment
of
Emergency Medicine, University of Oslo, Oslo, Norway; stian.ellefsen@hil.no Detection of
necrotic tissue after
ischemia is often done using 2,3,5-triphenyltetrazolium chloride (TTC).
However, tissue exposed to TTC is not suitable for molecular analyses,
and
alternative methods for infarct size assessment are warranted. Here we
report
an approach for estimation of infarct size using real-time RT PCR. In
short, an
external RNA control gene was added to infarcted heart tissue in
accordance
with a recently developed protocol, and was used for real-time RT PCR
normalization, giving “per wet-weight normalization”. The expression of
seven
conventional internal RNA control genes was investigated. Of these,
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was found to correlate
well
with TTC-measured infarct sizes, making it a potential infarct size
marker.
Indeed, we speculate that GAPDH expression may represent a more
reliable
measure of cell death than TTC. In addition, GAPDH stands out as the
most
suitable internal RNA control gene. In contrast, the measured amounts
of
ribosomal RNA (18S) and total RNA showed low correlation with infarct
size,
making them unsuitable for normalization of real-time RT PCR data. P141 Oxygen
restriction increases the infection potential of Listeria monocytogenes
-
verification of microarray chip data by quantitative real-time PCR Technical Listeria
monocytogenes has been
implicated in several food borne outbreaks as well as sporadic cases of
disease
during the last two decades. Increased understanding of the biology of
this
organism is important in the prevention of food borne listeriosis. This
is
highly relevant for safety assessment of this organism in food. We have
previously shown (Andersen
et al., BMC Microbiology; 2007, 7:55) that the environmental conditions
to
which L. monocytogenes is exposed prior to ingestion are decisive for
its in
vivo infective potential in the gastrointestinal tract after passage of
the
gastric barrier. Infection of Caco-2 cells revealed that Listeria
cultivated
under oxygen-restricted conditions were approximately 100 fold more
invasive
than similar cultures grown without oxygen restriction. This means that
not
only the number of Listeria present in a given food item, but that also
the
physiological condition of these bacteria is important for food safety.
The in
vitro and in vivo data suggest that an oxygen-restricted L.
monocytogenes cell
represents a significantly higher risk than a cell grown without oxygen
restriction. In order to
characterize genetic
differences contributing to different invasiveness, microarrray gene
chip
technology was applied to cDNA created from RNA isolated from oxygen
restricted
and non-restricted cultures. The analysis confirmed several relevant
genes to
be differentially regulated in the two environmental conditions e.g.
genes
related to virulence potential of Listeria monocytogenes. Quantitative
PCR was used to verify
the quantitative differences identified with the microarray chip for a
selection of relevant and differentially regulated genes. We will
present data,
which demonstrates successful verfication of the microarray chip data,
both
regarding technical as well as biological replicates. P142 Protein
Thermal Shift Assays Using Applied Biosystems Real Time PCR Instruments 1Applied
Biosystems, part of Life Technologies, CA, United States of America; 2IMAGIF,
Bât 23B, ICSN-CNRS, Gif-sur-Yvette, France; madeline.odonoghue@appliedbiosystems.com
The Protein
Thermal Shift Assay
(TSA) is a rapid and sensitive tool for monitoring protein
thermostability;
aiding in the identification of optimal conditions or
conformations/sequences
that favour protein stability, including the investigation of
protein-ligand
interactions. TSA is based on temperature-induced protein denaturation,
monitored using an environmentally sensitive dye, such as SYPRO Orange,
that is
naturally quenched in a stable, aqueous solution. As the temperature
increases,
the protein will start to denature, exposing hydrophobic regions that
will bind
the SYPRO Orange dye, leading to a proportional increase in
fluorescence. The
increase in fluorescence over time is measured using a real-time PCR
instrument. The inflection points of the resulting
fluorescence/temperature
plot are used to compare different test conditions, and the extent of
the
observed temperature shift is considered to be proportional to the
affinity of
a ligand to a specific protein, or representative of the stability of
the
protein in certain solution conditions. TSA data have been obtained
from the
whole range of Applied Biosystems Real Time PCR Systems; including the
7900 HT,
7500 Fast and StepOnePlusTM Real Time PCR Instruments, demonstrating
the
versatility of these systems. TSA has wide-ranging applications, and
the use of
96- or 384-well plates lends this assay to high-throughput screening
for
unknown ligands and protein inhibitors in compound libraries,
protein-substrate
interactions, concentration-dependent stabilisation conditions, and
mutational
screening of protein sequences for optimization of binding stability.
In
addition, TSA benefits protein crystallography studies, for example, in
determining the concentration of a compound that will provide maximal
stability
in order for a protein to reach maximum occupancy. The benefits of
performing a
TSA with an Applied Biosystems Real Time PCR System include the
flexibility of
run-method programs, catering for a range of data resolution
requirements and in
the use of small reaction volumes, providing fast and accurate results
with
only a few µg of protein. P143 QRT-
PCR in translational oncology iau of
tonekabon, Quantitative
real-time PCR (qrt-PCR)
technology has recently reached a level of sensitivity, accuracy and
practical
ease that supports its use as a routine bioinstrumentation for gene
level
measurement. Analysis of transcriptional activity of tumor cells or
detection
of tumor markers by qrt-PCR has the potential to change lung cancer
diagnosis
and treatment. Quantitative RT-PCR is characterized by unparalleled
sensitivity
and specificity, with very reliable reproducibility. Its prime
advantage for
gene expression analysis is its broad dynamic range of 107-fold.
Moreover, it
is cost-effective, feasible in every day laboratory routine and
efficient in
terms of biological material consumption.Knowledge of the biochemical
principles underlying this biotechnology can be of great value to
interpret
correctly qrt-PCR data. P144 Real-time
PCR assays for Pseudo-nitzschia species in Irish waters 1Molecular
Diagnostics Research Group, National University of Ireland Galway,
Galway,
Ireland; 2Martin Ryan Institute, National University of
Ireland
Galway, Galway; eibhlin.nicheidigh@nuigalway.ie
Pseudo-nitzschia
is
a genus of marine planktonic diatoms. Some Pseudo-nitzschia species
have
the ability to produce domoic acid (DA), a toxin which can accumulate
in
filter-feeding shellfish tissue and cause amnesic shellfish poisoning
in humans
via the food chain. Monitoring Irish waters for the Pseudo-nitzschia
genus
is traditionally performed by light microscopy but this method cannot
routinely
identify Pseudo-nitzschia to species level. Species
identification requires
sophisticated electron microscopy which is an unsuitable technique for
monitoring. Molecular methods, including real-time PCR, have the
potential to
rapidly identify Pseudo-nitzschia to species level. We have
developed real-time PCR
assays targeting the rRNA ITS1 region for the rapid, high-throughput
detection
and identification of two Pseudo-nitzschia species - P.
multiseries and P. seriata . These tests incorporate
hybridization probes
and are
performed on the Roche ™ LightCycler. Specificity of each test has been
verified using a broad panel of phytoplankton species and the limit of
detection of each test is 10 cells in a 25 ml water sample. The
real-time PCR
tests have been successfully used to identify P. multiseries and
P.
seriata in field samples from Irish shellfish production waters.
The
application of these assays for quantitative detection of P.
multiseries and P. seriata in field samples is under
investigation. P145 Sensitive
Detection of KIT D816 in Patients with Gastrointestinal Stromal Tumours
(GIST).
Implication for Molecular Diagnostics and Pharmacogenomics 1Italian
Association of Pharmacogenomics and Molecular Diagnostics, Italy; 2Clinical
Pathology Lab "clinica Villa dei Fiori", Naples, Italy; 3Servizio
Immunotrasfusionale P.O. "S. G. Bosco", Naples, Italy; 43Department
of Physiopathology, Clinical Biochemistry Unit, University of Florence,
Italy.; rdifrancia@iapharmagen.org
Background:
The pathogenic variation
at codon 816 of exon 17 (D816V) in the KIT gene, occurring in some
Gastrointestinal Stromal Tumours (GISTs), leads to constitutive
activation of
tyrosine kinase activity and confers resistance to the tyrosine kinase
inhibitors, like imatinib mesylate. The molecular
recognition of this
mutation, in patients with GIST, is important for determining treatment
strategy but, because the ratio between malignant cells, carrying this
genetic
variation, and normal cell population is often small, standard
detection
methods can be unsuccessful. Methods: We developed high sensitivity
method
based on Locked Nucleic Acid (LNA) oligomers for selective detection of
2447
A>T D816V mutation of KIT gene. LNAs have been used to enhance
mutant allele
detection in a large excess of wild type allele, from DNA extracted by
formalin-fixed paraffin-embedded of 19 GIST’s biopsy. In addition we
compared
this result with those obtained by direct sequencing . Results: The
assay sensitivity of
the method was 0.5%, assessed by serial dilution of DNA extracted from
HMC-1
cell line (carrying D816V mutation), mixed up wild type DNA. By LNA
assay the
57,9% (11/19) of patients were identified as carriers of mutation at
codon 816,
while direct sequencing was able to detect the mutation in only 2/19
(10.5%)
patients. In addition, LNA assay combined to pyrosequencing platform,
were able
to discriminated 8 D816V point mutation in exon 17 and 3 additional
pathogenic
variations in short context sequence D816Y, D816N and N822Y of the 11
positive
cases. Conclusion: These findings demonstrate that the LNA assay
combined to
Pyrosequencing is an highly sensitive and well discriminating approach
for
detection of KIT mutation at codon D816 and codon N822 in patients with
GIST. P146 Detection
of nucleophosmin (NPM1) gene mutations with different methods in
patients with
AML University
Medical Centre Ljubljana,
Slovenia; marija.mandelc@kclj.si
A
nucleophosmin (NPM1) gene encodes
nuclear multifunctional proteins. Mutations – insertions of the 4 bp at
the
exon 12 of the NPM1 gene, located on chromosome 5q35 are the most
prevalent in
adult patients with acute myeloid leukemia (AML) and normal karyotype.
Patients
positive for mutations in NPM1 gene and negative for internal tandem
duplication
mutations in FMS-like tyrosine kinase 3 (FLT3) gene have favourable
prognosis
in this group1-4. The aim of
this study was to compare
the results of the NPM1 gene mutations detection by PCR-gel detection
and
sequencing of the amplified PCR products. Furthermore, we want to
introduce the
real-time quantitative polymerase chain reaction (RQ-PCR) assay for
quantitative assessment of the most frequently observed mutations (type
A and
B)1. The diagnosis
of AML was established
followed World Health Organization classification. 95 patients with AML
were
included in the study. Bone marrow aspirates were used for the
isolation of
mononuclear cells (MC) by ficoll density centrifugation. RNA was
isolated from
MC by High Pure RNA Reagent Kit (Roche) and cDNA was performed by
SuperScript
II reverse transcriptase (Invitrogen). Amplification of the exon 12 of
the NPM1
gene was performed with the forward and reverse primer as described3.
The PCR
products were visualized after agarose gel (4%) electrophoresis by
ethidium bromide
staining. All PCR- amplified samples were purified and sequenced.
RQ-PCR
reaction assay for type A and B mutation were performed with
MutaQuantTM
Standards Kit (Cancer Profiler, Ipsogen)1. The
concordance between PCR-gel and
sequence detection was 100%. NPM1 mutations were identified in 22 (23%)
of the
95 AML patients. The most common detected mutation type was insertion
of the
TCTG tetranucleotide (type A, 77%), followed by insertion of the CATG
(type B,
9%), CGTG (type C, 9%), CCTG (type D, 5%) and CCAG (type NM, 5%). These
are in
agreement with recently published studies 1-4. Results for mutation
type A and
B obtained with RQ-PCR assay were in agreement with results obtained
with of
PCR-gel and sequence detection. At RQ-PCR reaction assay we also
performed
inter and intra assay for reproducibility. According to this assay
variation
coefficient value was below 30%, which is in agreement with literature
data. Our study
shows, that PCR-gel
detection is suitable screening method for identification of NPM1
mutations. A
sequence detection assay is suitable for confirmation of presence
mutations.
RQ-PCR was found out as sensitive and reliable method. On the basis of
variation coefficient was also found out that RQ-PCR method is suitable
for
monitoring minimal disease in AML patients. 1 Gorello P,
et al. Leukemia, 2006;
20: 1103-1108. 2 Verhaak
RGW, et al. Blood, 2005;
106: 3747-3754. 3 Döhner
K, et al. Blood, 2005; 106:
3740-3746. 4 Schlenk RF,
et al. NJEM, 2008;
358: 1909-191 P147 Multiplex
qPCR for influenza diagnostics 1Robert
Koch Institute, NRZ Influenza, FG12 „Virale Infektionen“, Berlin,
Germany; 2Robert
Koch Institute, Zentrum für Biologische Sicherheit, Berlin,
Germany; biereb@rki.de The To minimize
hands-on time and
financial costs, we established a multiplex real-time PCR assay for the
parallel
detection of Influenza A, Influenza B and an internal control (IC).
Multiplex
real-time PCRs were established for further differentiation of HA (H1,
H3) and
NA subtypes (N1, N2) of human influenza viruses. Furthermore, a
multiplex
real-time assay was also developed for the specific detection of
important
subtypes of avian influenza viruses (H5, H7, H9). All multiplex PCRs
have the
same specificity and detection limit as the uniplex PCRs, even though
the
fluorescence intensities of separate probes in the multiplex PCR are
decreased
in comparison to uniplex PCRs. Moreover, duplex infections only have a
negligible effect on the detection limit. The PCRs were evaluated with
patient
specimens from the influenza seasons 2005/2006 and 2006/2007. The results
of our study confirm
that multiplex PCR is a reliable tool for the detection of respiratory
pathogens and, therefore, especially suitable for laboratories with
high sample
throughput. P148 Detection
of defective qPCR samples with Outlier of Kineret software Outlier is a test
tool implemented within the Kineret software for real-time PCR
data analysis. The aim of the test is to identify PCR samples with
defective
amplification. By finding and removing such samples from the analysis, Outlier improves the overall results.
The computational algorithm behind the Outlier is different from
recently
published methods based on comparison of amplification efficiencies of
samples.
The unique patented method employed by Outlier
utilises the principle of simultaneous recognition of several geometric
characteristics, similar to that applied, for example, for the
detection of
fake coins in vending machines. The recognition of defective samples is
based
on a comparison to a reference set of well performing samples, usually
the
standard curve samples. The reference set is self-assed for its
homogeneity
prior to its use, and possibly itself corrected by excluding
incompatible
samples. Alternatively, where no prior assumption of good performance
can be
imposed on subset of samples, all samples are taken as a reference. We
achieved
superior performance in terms of artificially inhibited samples
retrieved by
the Outlier as compared to recently published methods. please direct your enquiry to Martina.Reiter@bioeps.com or qPCR2009@wzw.tum.de © 2009
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