qPCR 2007 Symposium POSTER Presentations

Abstracts - Poster presentations


Main Session:   microRNA – siRNA Applications

Poster number   P 001    P 009

Location:           Student Cafeteria

P 001

Regulation of cadmium-induced responses in Arabidopsis thaliana: The role of microRNAs.

Smeets K., Cuypers A., Donckers K., Remans T., Vangronsveld J.

Centre for environmental sciences, Hasselt University, Belgium

Email: karen.smeets@uhasselt.be

As a highly toxic metal, knowledge about cadmium-induced pathways in plants is rather scarce. Cadmium does appear to provoke several oxidative stress related effects, similar to other heavy metals. Because of its non-redox active nature, the induced redox disequilibrium has to be established via indirect pathways. A specific role of this increased ROS-content in the regulation in cadmium-induced responses, however, remains to be elucidated.

Therefore, the early effects of cadmium toxicity were examined by performing a complete transcriptome analysis in Arabidopsis thaliana in combination with the analysis of specific cadmium-induced microRNAs. From these results, a highly coordinated expression response can be concluded during moderate cadmium toxicity, in which an increased ROS-content could play a central role. Furthermore, the induced/inhibited regulative genes are already responding after a few hours of exposure and expression is well regulated between the different organs.

In this study environmental realistic exposure concentrations were used, therefore the outcome is highly relevant for further research on heavy metal contamination.

P 002

Efficient and specific quantification of mammalian microRNAs using a novel real-time PCR approach.

Martin Kreutz1, James Qin1, Holger Engel 2, Po-Jen Shih1, Martin Schlumpberger 2, Subrahmanyam Yerramilli1, and Eric Lader1

1QIAGEN Sciences, Germantown, USA and 2QIAGEN GmbH, Hilden, Germany

Email: martin.schlumpberger@qiagen.com

We have developed an efficient and accurate method for transcriptome-wide miRNA quantification using a SYBR Green based, real-time PCR detection system. The miScript System is highly specific and sensitive, and requires very small amounts of input RNA. The system enables detection of miRNAs as well as mRNAs using the same cDNA preparation allowing simultaneous quantification of miRNA and target mRNA.

A single cDNA prep is sufficient for quantification of several miRNAs of interest, avoiding the need to prepare multiple cDNA preps to quantify each miRNA. Use of a single prep eliminates potential variation that could arise during multiple cDNA synthesis reactions. We will demonstrate the application of this technology to miRNA expression profiling in a model human cell-culture system.

This technology offers researchers a sensitive, specific, and easy-to-perform approach for accurate expression profiling of miRNAs using a small amount of total RNA containing miRNAs. A single cDNA prep from a precious RNA sample is sufficient for profiling of all the known miRNAs in a given model system. This is a substantial advance in the state of the art and would be of broad interest to all scientists studying miRNAs and their targets

P 003

Measure of miRNAs in microdissected colorectal tumor tissues: optimization of RNA preparation.

Stefania Gelmini, Marta Tarter, Francesca Salvianti, Claudio Orlando, Lisa Simi, Mario Pazzagli, Pamela Pinzani.

Clinical Biochemistry Unit, Dept. of Clinical Physiopathology, University of Florence, Italy

Email: s.gelmini@dfc.unifi.it

MicroRNAs (miRNAs) are short non-coding RNA molecules involved in gene expression regulation by repressing translation or cleaving RNA transcripts. Recently a connection between miRNAs and cancer was suggested: several authors reported an altered expression of miRNAs in different cancers where they could function as both tumor suppressor or oncogenes. miRNA analysis has generally been performed by microarray techniques in order to identify the specific expression profile involved in different types of cancers. On the basis on known complementary nucleotide sequences, computer-based prediction model have been developed in order to identify the specific target genes of miRNA; several relevant association with target genes, either oncogenes or tumor suppressors, have been found but the genetic pathways under miRNA regulation have still largely to be determined The laser microdissection is the method of choice to collect pure cell population starting from complex tissues. This technique has been already largely used to identify specific expression profile of mRNA in a single cell or in different cell compartment in the same tissue. In order to obtain in a series of microdissected colorectal tumor tissue samples a sufficient quantity of RNA for gene expression analysis and for a specific related miRNA quantification, we have utilized several RNA extraction methods either specific for miRNA (MIRACLE miRNA Isolation kit, Stratagene) or for total RNA by a specific reagent (Trizol, Invitrogen) or by a lysis buffer (Side Step Lysis and Stabilisation Buffer, Stratagene). We have performed a quantitative relative measure of total (18S) and miRNA by Real Time PCR (TaqMan); the miRNA were miR-31 and miR-135-b. These two miRNAs resulted altered in colorectal tumors in comparison to relative normal tissues (Bandres et al, 2006). The relative quantification is obtained normalising to a RNU6b miRNA. All miRNA measurements were performed by TaqMan MicroRNA assay (Applied Biosystem). For the evaluation of total RNA extracted we have used a primers and probe for 18S RNA (Pre-Developed TaqMan Assay Reagents, Applied Biosystem). Results obtained by MIRACLE show high specificity in the miRNAs extraction and high accuracy for their detection starting from about 10 cells/tube. The other techniques (Trizol,Lysis Buffer Stratagene) were able to detect both total RNA and miRNAs with sufficient sensitivity, reproducibility and accuracy, even if the performances of Stratagene buffer was affected, in same cases, by sample interferences due to the presence of DNA or proteins in the lysate. In all samples (n= 5), miR-31 and miR-135-b resulted overexpressed in tumor microdissected samples in comparison in normal paired tissue.

P 004

MicroRNA Expression Signatures as potential Biomarkers in Thyroid Cancer.

Astrid Potratz1, Cara Martin2,3, Simone Guenther1,O’Leary J.23, Orla Sheils2

(1)Applied Biosystems, Applera Deutschland GmbH, Frankfurter Strasse 129b, Darmstadt, Germany; (2)Histopathology, Trinity College, Dublin, Dublin, Ireland; (3)Pathology, Coombe Women's Hospital, Dublin 8, Ireland

Email: GuenthSM@eur.appliedbiosystems.com

Cancer is caused by abnormal cellular proliferation and the inappropriate survival of damaged cells, which may result in tumor formation. Cells have developed several safeguards to ensure a correct and coordinated cell division, differentiation and death. A defect in the regulatory factors, e.g. tumour-suppressor genes and oncogenes, will increase the propability of tumour development.

MicroRNAs (miRNA), short non-coding, single-stranded RNAs, constitute a novel class of negative gene regulators. Specific miRNAs may even control several target mRNAs resulting in diverse and complex biological processes. Recent evidence indicates that miRNAs might also function as physiological tumour suppressors and oncogenes. Repressing the expression of important cancer-related genes, miRNAs might therefore prove a valuable tool in the diagnosis and treatment of cancer.

Thyroid cancer cell line: The expression profiles of 157 miRNAs in thyroid cancer cell lines have been analyzed using the Applied Biosystems TaqMan MicroRNA Assays. Specific miRNA patterns associated with different pathological pathways have been identified, indicating that miRNAs can be used to reliably discriminate between two common triggers of thyroid cancer. The predicted mRNA targets of these differentially expressed miRNAs belonged to genes involved in regulatory molecular functions such as signalling pathways, cell division control and transcription. This correlates nicely with the respective gene expression profiles using the Applied Biosystems Whole Genome Array System. There, genes involved in the MAPK signalling pathway, oncogenesis and transcriptional regulation where shown to be upregulated whereas several cell cycle regulators were found to be down-regulated.

Cervical cancer cell line: The expression profile of 180 miRNAs in two cervical cancer cell lines was studied using TaqMan miRNA Assays. miRNA was extracted using Ambions mirVana miRNA isolation system. miRNA extracted from histologically normal cervical tissue was used as a reference.

A specific different miRNA expression signature in the cancer cell lines was observed compared to normal cervical tissue. Several of these differentially expressed miRNAs are predicted to interact with cell cycle regulatory molecules. These findings highlight the potential importance of miRNA molecules also in cervical cancer.

The determination of miRNA profiles as a new class of biomarkers has the potential to significantly improve diagnostic accuracy and prognostic information.

P 005

Effects of segmental trisomy on the expression of microRNA genes by qRT PCR.

Blatny R., Ivanek R. & Forejt J.

Institute of Molecular Genetics AS CR, Prague, Czech Republic

Email: blatny@biomed.cas.cz

In our pilot study, we have used adult mice of the Ts43H mouse model of human aneuploidy syndromes carrying largest known segmental trisomy of an autosome with more than 300 genes [1]. We were interested in consequences of the segmental gene dosage imbalance on transcription of genes located in the trisomic region (up to 21 genes, depending on the tissue type) in comparison with genes located in the disomic region (up to 15 genes) of the same chromosome.

MicroRNA genes are known to be example of non-codingRNA genes with strong regulatory potential and are therefore candidate genes in study of development of different pathological states including aneuploidy syndromes. We have measured expression levels of three mature microRNA molecules located in a cluster inside of the trisomic region and one mature microRNA located on another chromosome. We have selected brain as our target tissue, since cognitive abilities were shown to be affected in the model [1], and we used liver as a control tissue.

We are reporting significant differences in individual protein-coding genes between control animals and trisomic animals, mainly for genes in the trisomic region. The average expression level of protein-coding genes in the trisomic region was ~1.6-fold in both liver and brain, which corresponds with the altered gene dosage and does not indicate any kind of dosage compensation. In the disomic region, the average expression level was ~1.0-fold in liver and ~0.9-fold in brain, which indicates slight downregulation of the disomic part in brain. However, statistical significance of this difference remains unclear. These findings are generally in agreement with studies on different mammalian models of aneuploidy and contrast with non-additive gene expression reported in some plants and Drosophila melanogaster .

Finally, we are presenting for the first time measurements of gene-dosage effects on microRNA genes in a mammalian genome. The three microRNA genes located in the trisomic region were upregulated ~1.5-fold. However, the microRNA gene located on another chromosome was found downregulated (0.8-fold).

1. Vacik, T., et al., Segmental trisomy of chromosome 17: A mouse model of human aneuploidy syndromes. PNAS, 2005. 102(12): p. 4500-4505.

P 006

MicroRNA profiling of breast cancer using Locked Nucleic Acid (LNA) based technologies.

Jacobsen N.1, Nørholm M.1, Glue C.1, Stahlberg N.1, Eriksen J.2, Svane I. M.2, Flyger H.2, Balslev E.2, Møller S.1, and Litman T.1.

1Exiqon, Vedbaek, Denmark and 2Herlev University Hospital, Herlev, Denmark

Email: jacobsen@exiqon.com

Abnormal expression of microRNAs (miRNAs) in cancer implies that these small ~22-nucleotide molecules play a role in oncogenesis Therefore miRNAs may comprise a novel class of diagnostic and prognostic signatures. Here, we study the global expression profiles of miRNAs in breast cancer and normal adjacent tissue in order to identify possible new biomarkers for breast cancer.

Here, we present miRNA expression profiles from tumor and normal breast tissue, and found numerous differentially expressed miRNAs, including those previously reported to be associated with breast cancer, such as let-7a/d/f, miR-125a/b, miR-21, miR-32, and miR-136. The differential expression profiles from miRCURYTM LNA Arrays have been confirmed using real-time PCR assays. The real-time RT-PCR detection is a useful tool in addition to Northern blot analysis.

We envision that the different platforms compared in the current study will facilitate an efficient workflow for identification of e.g. disease related miRNAs and subsequent development of reliable diagnostic and prognostic assays.

P 007

Comparison of miRNA expression patterns using the total RNA extracted from formalin-fixed paraffin-embedded (FFPE) cells and that extracted from snap frozen.

Li J.1, Smyth P. 1, Flavin R.1, Cahill S. 1, Denning K. 1, Aherne S. 1 Guenther S. 2, O’Leary J. 1, Sheils O1

(1)Department of Histopathology, Institute of Molecular Medicine, St James’s Hospital, Dublin, Ireland. (2)Applied Biosystems, Darmstadt, Germany

Email: GuenthSM@eur.appliedbiosystems.com


Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an abundant source of clinical specimens; however they have limited utility in applications involving analysis of gene expression due to mRNA degradation and modification during fixation and processing. Interestingly, miRNAs are a small class of RNA recently described as playing important roles in gene regulation, yet their robustness in FFPE is largely unknown. This study analyzed 160 miRNAs in paired snap frozen and FFPE cells to investigate if miRNAs may be successfully deteced in archival specimens.


N-thy-ori cells were grown to confluence and aliquots with equal cell numbers were (a) snap frozen and (b) formalin fixed and paraffin embedded into a cell block. Total RNA was extracted using protocols: (a)Ambion mirVana miRNA Isolation kit for snap frozen cells, (b)Ambion RecoverAll Total Nucleic Acid Isolation Kit for FFPE cells. The quality and quantity of RNA yields was measured with Nanodrop and TaqMan® microRNA assays, Human Panel-Early Access Kit.


To achieve 50 ng of total RNA for each RT reaction, 10,000 ng of total RNA (for 200 assays), was extracted from approximately 2x106 FFPE cells and 1.7x105 snap frozen cells. TaqMan® analysis showed a good correlation of miRNA expression pattern between FFPE and snap frozen cells, with R2>0.95. The mean of ΔCts (Cts_FFPE normalized to Cts_snapfrozen) was -1.04107 and the median was -1.152 with p<0.0001. 65.58% of ΔΔCts (ΔCts normalized to ΔCts_mean), 101 out of 154 determined assays, were between +1 and -1. There was some outlying data in performing the comparison between snap frozen and FFPE cells, most notably miR-146 exhibited ~20 fold decreased expression and miR-302b* ~8 fold increased expression.


miRNA extracted from FFPE blocks was successfully amplified using Q-RT-PCR. The levels of expression of miRNA detected in total RNA extracted from FFPE were higher than that extracted from snap frozen cells when the amounts of total RNA were identical. It seems reasonable to conclude that this phenomenon was most likely caused by methylol cross-links between RNA and protein resulting small RNA molecules being less compromised than their larger counterparts. The majority of miRNAs demonstrated reliable expression levels in FFPE compared with snap frozen paired samples suggesting these molecules might prove to be robust targets amenable to detection in archival material in the molecular pathology setting.

P 008

A new microfluidic Assay for the Analysis of small RNAs.

Marcus Gassmann1 , Hans-Joachim Mollenkopf2, Marc Valer3, Martin Greiner1

1Agilent Technologies, Waldbronn, Germany, 2Max-Planck Institute for Infection Biology, Berlin, Germany and 3Agilent Technologies Inc., Santa Clara, CA, USA

Email: marcus_gassmann@agilent.com

MicroRNAs (miRNAs) are short 20-22-nucleotide RNA molecules that have been identified recently as sequence-specific regulators of many cellular processes such as apoptosis, proliferation and differentiation. Meanwhile hundreds of microRNAs have been discovered in the genomes of animals and plants, but they are only beginning to be classified by their functional roles. One of the major drawbacks is the lack of adequate analytical methods for the analysis of small RNA samples.

Here we describe a Lab-on-a-Chip based assay that is able to perform very sensitive high resolution analyses of small RNA samples on the Agilent 2100 Bioanalyzer instrument. The assay delivers information about integrity, size and concentration of small RNA species. Purified or enriched small RNA fractions, as well as total RNA samples in concentrations can be run in concentrations down to 100 pg/µl.

The Agilent 2100 Bioanalyzer is a microfluidic instrument platform designed for fast separation and quantification of DNA, RNA, and proteins as well as flow cytometric analysis on cells.

P 009

Multiplex microRNA TaqMan® Low Density Array: A High-Throughput Screening Tool for miRNA Profiling.

Yulei Wang, Raymond Samaha

Applied Biosystems, United States of America

Email: wangyy@appliedbiosystems.com

<>MicroRNAs (miRNAs) are an abundant class of endogenous ~ 22-nucleotide (nt) RNAs that have been shown to have critical functions in a wide variety of biological processes, including development, cell proliferation and death, and oncogenesis. Because of their short size and the sequence similarity between family members, developing a genome wide profiling approach that is sensitive and specific is rather challenging. miRNA arrays as well as bead-based approaches have been used for miRNA profiling, however, their detection sensitivity and specificity are typically low. To address these issues we have developed miRNA TaqMan Low Density Arrays (miRNA TLDA), in which highly sensitive and specific miRNA TaqMan® assays targeting 369 individual human miRNAs and 16 endogenous controls are pre-loaded into a 384-well micro fluidic card. The reverse transcription step (RT) can now be performed in a 48-plex fashion and the resulting cDNA loaded into the miRNA TLDA for real-time PCR detection without the need for robotics. We will be presenting applications of this technology in screening biomarkers in clinical samples and cancer cell lines.


Main Session:   Single Cell qPCR

Poster number   P 010    P 011

Location:           Student Cafeteria

P 010

Profiling of the TrpC expression pattern in cerebellar Purkinje cells by quantitative single-cell RT-PCR.

Dragicevic E.1,Blum R.2, Hartmann J.1, and Konnerth A.1

1Friedrich-Schiedel Institute of Neuroscience, TU Munich and 2Physiological Institute, Physiological Genomics, LMU Munich

Email: elena.dragicevic@lrz.tu-muenchen.de

The TRPC (Transient Receptor Potential Canonical) cation channel family consists of seven members; TRPC1-7 (Clapham et al. 2001). TRPC subunits are expressed in many brain areas but little is known about their cellular function in neurons. It has been reported that the TRPC1 cation channel is involved in the mGluR1-dependent slow excitatory postsynaptic current (ImGluR) in Purkinje cells (Kim et al., 2003). However, we found that ImGluR can be evoked in TRPC1 knock-out mice. This suggests a role for other TRPC family members in the activation of ImGluR.

To understand the molecular basis of TRPC-mediated postsynaptic currents, we investigated the cell type-specific gene expression patterns and levels of TRPC encoding transcripts in single Purkinje cells of the cerebellum using a quantitative Reverse Transcriptase-PCR (RT-PCR) approach (Durand et al. 2006). In our experiments, single Purkinje cell somata were obtained by whole soma suction from acute cerebellar slices, using microcapillaries. RT-reactions, followed by purification of single cell cDNA material were amplified in a real time PCR device (Lightcycler). Quantification was performed by using high-resolution standard curves, with comparable efficiencies, for each subunit. In parallel, single cell RT-reactions were validated by quantification of the house-keeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) in a second qPCR reaction using 1/10 amount of the single-cell cDNA. In addition, we use specific TRPC knock-out mutants to correlate the expression levels of TRPC subunits with the corresponding physiological phenotypes in Purkinje cells.

Our findings revealed that the subunit TRPC3 (372 copies/cell) and not TRPC1 (44 copies/cell) is the predominant TRP-family member in Purkinje cells of wild type mice. TRPC4 (10 copies/cell), TRPC6 (4 copies/cell), and TRPC7 (5 copies/cell) have been detected in few copies, while TRPC5 was not detected yet. TRPC1 knock-out mice show the same TRPC subunit expression pattern. The electrophysiological analysis as well as the quantitative single cell RT-PCR experiments are leading to the conclusion that TRPC3 and not TRPC1 is the most likely molecular candidate for ImGluR in cerebellar Purkinje cells of the mouse.

Clapham, D. E., Runnels, L. W., Strubing, C. (2001) The TRP ion channel family. Nat Rev Neurosci 387-96.

Kim, S. J., Kim, Y. S., Yuan, J. P., Petralia, R. S., Worley, P. F., Linden, D. J. (2003) Activation of the TRPC1 cation channel by metabotropic glutamate receptor mGluR1. Nature 426:285-291.

Durand, Marandi, Herberger, Blum & Konnerth (2006) Pflügers Arch. Eur J. of Physiol. 451, 716-726.

P 011

Changes in the gene expression of mRNA transcripts for insulin like growth factor (IGF-I), their receptor (IGF-IR) and facilitative glucose transporter (Glut-I) in IVM oocytes and preimplantation embryos of Buffalo derived from Somatic Cell Nuclear Transfection.

S.C. Gupta, Neelam Gupta, Alok Pandey

National Bureau of Animal Genetic Resources, Karnal, India

Email: guptasc@yahoo.com

For low rate of SCNT derived cloned embryos reaching to normal and viable clones, one hypothesis is that transcription of one or several developmentally important genes is affected by the in vitro environment possibly leading to the disturbance of process of differentiation and organogenesis. Therefore embryos exhibiting abnormal expression of embryonic genes may be an early indication of incomplete reprogramming that could result in lower survival rates. The real-time quantitative polymerase chain reaction (rtqPCR) has overcome the limitations of conventional, time consuming quantitative PCR strategies and has been matured into a routine tool to quantify gene expression levels, following reverse transcription (RT) of mRNA into complementary DNA (cDNA). It is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos. One of the areas of interest in this regard, is the analysis of gene expression patterns in nuclear transfer (NT) embryos to dissect the processes that failed and develop means to overcome the limitations imposed by these factors. The main objective of this study was to develop an easy and rapid method for measuring gene expression in a single cell by real-time PCR without RNA extraction and purification by using cell to cDNA II kit (Ambion). Based on the developed RT-PCR methodology, we constructed cDNA libraries with single matured oocytes at 0h, 6h, 12h, 18h and 24h of maturation and of embryos at different stages of development (2-cell, 4-cell, 8-cell, 16-cell, morula and blastocyst). Real time PCR cycler was performed using Brilliant SYBR Green Q-PCR master mix (Stratagene) to determine more precisely the transcription levels of IGF-I, IGF-II, and Glut-I in SCNT produced buffalo embryos derived from skin fibroblast cells at preimplantation development. The primers of selected genes GAPDH (reference) and IGF-I, IGF-II, and Glut-I (target genes) for expression studies were designed using the Gene Bank sequences within only one exon in the range of 80-200 bp products. Standard curve for reference and all the target genes were developed for the relative quantification of gene expression. IGF-I transcript expression was clearly visible only in matured oocytes. It was elevated up through 12h of the culture and then declined gradually at the end of 24hrs maturation. IGF-IR, was expressed throughout preimplantation development upto the blastocyst stage, while the expression of facilitative glucose transporter (Glut-I) was also expressed in all stages studied. Gene expression analysis of those genes which plays a important role in activation and reprogramming of matured oocytes and cloned embryos would enable us to better understand the early biological events during preimplantation after nuclear transfer.

Session:   Immuno - qPCR

Poster number   P 012    P 013

Location:           Student Cafeteria

P 012

Real-time immuno-PCR: a new perspective for prion blood screening tests.

Ruelle V., ElMoualij B., Heinen E. and Zorzi W.

Center of research on Prion Proteins, University of Liège, 4000, Belgium

Email: v.ruelle@ulg.ac.be

Prion diseases such as Creutzfeldt-Jakob of human, scrapie of sheep and bovine spongiform encephalopathy of cattle are fatal neurodegenerative disorders characterized by behavioural and locomotor changes, cerebral amyloid plaques and spongiform degeneration of the brain1. In 1996, the first case of variant of Creutzfeldt-Jakob disease (vCJD) was diagnosed in the United Kingdom, through the consumption of prion-contaminated meat and borne meal with Bovine Spongiform Encephalopathy (BSE). Since, their number increased whereas cases of BSE were declining. Until the end of January 2007, 158 people were death from vCJD and 7 are still alive2. The potential for transmission of vCJD by blood transfusions was demonstrated experimentally3 prior the apparition of apparent cases of human transmission of vCJD by transfusion4. From December 2004, 3 people are already death following a transfusion of contaminated blood.

The classical techniques used to diagnosed prion protein (Western blot assays and enzyme-linked immunosorbent assays)5 are not sufficiently sensitive to detect the low levels of prion in the blood. Consequently, it is necessary to develop a sensitive technique allowing the diagnosis of prion protein in blood.

In this study, we therefore focussed on the detection of the prion protein in blood by immuno-quantitative-PCR (iqPCR)6. This technique combines the sensitivity of PCR, by an exponential amplification of reporter DNA, and the specificity of the detection of antigens, by antibodies in an ELISA format7-8. To illustrate the advantages of iqPCR, we have compared it with a conventional ELISA technique in experiments aimed at detecting the resistant form of prion protein in human plasma. Using iqPCR, a minute quantity of prion was detected in blood spiked with infected CJD sample with a detection threshold at least 400-fold lower than classical ELISA.

The iqPCR technique being ultra-sensitive, it could be a technique of choice for the development a new blood screening tests allowing the prion protein diagnosis in infected human and animals ; both at ante-mortem and post-mortem stage.

P 013

TSS-mediating staphylococcal toxins: A novel quantitative real-time immuno-PCR approach for ultra-sensitive detection of SEB and TSST-1.

Andreas Fischer1, Thorsten Kuczius2, Christof von Eiff1, Georg Peters1, Karsten Becker1

University Hospital of Münster, Institute of Medical Microbiology, Germany(1) University Hospital of Münster, Institute of Hygiene, Germany(2)

Email: a.fischer@uni-muenster.de

TSS-mediating staphylococcal toxins: A novel quantitative real-time immuno-PCR approach for ultra-sensitive detection of SEB and TSST-1

Staphylococcus aureus is a major human pathogen characterized by a strain-dependent spectrum of virulence factors. One of these is the family of the bacterial pyrogenic toxin superantigens (PTSAg), comprising the toxic shock syndrome (TSS) mediating toxins TSST 1 and staphylococcal enterotoxin B (SEB). As morbidity and mortality from TSS are substantial, early and reliable recognition of TSS is critical. In addition to their nature as superantigens, enterotoxins also function as potent gastrointestinal toxins with a major public health impact.

Immunological methods used until now are known to be limited in sensitivity and specificity, revealing an obvious need for new highly sensitive and specific methods for the detection of staphylococcal toxins. For this reason, two quantitative real time immuno-PCR (qRT-iPCR) approaches for the detection of SEB and TSST-1 have been developed.

The detection of TSST-1 and SEB, respectively, was achieved by coating toxin-specific polyclonal sheep antibodies (ABs) to microtiter plates in order to capture the target superantigens followed by specific detection of the antigen-AB complex. The resulting immunocomplex was subsequently detected using a covalent antibody-DNA complex, which was synthesized using amino-modified and maleimide-activated reporter DNA and N-succinimidyl-S-actyl-thioacetate-modified secondary detection antibodies. Quantitative real-time amplification of the reporter-DNA was performed for final detection of the toxins.

By usage of this qRT-iPCR technique, superantigen toxin was highly reproducible detected at approximately 10 to 100 pg/ml (0,4 to 4 amol/µl), thereby lowering the limit of detection (LOD) of these toxins by a factor of up to 100 compared to commercially available EIAs. Furthermore, qRT-iPCR offers a high versatility, giving the opportunity of adapting the protocol to detect a broad range of antigens, provided that antibodies for the desired antigens are available. Covalent attachment of different reporter-DNAs to specific antibodies could be used to extend the qRT-iPCR to a multiplex detection platform.

Session:   Pre-analytical-Steps

Poster number   P 014    P 022

Location:           Student Cafeteria

P 014

Evaluation of different RNA extraction methods for small quantities tissue from the marine flatworm Macrostomum lignano.

Plusquin M., Smeets K., Geerdens E., Remans T., Cuypers A., Artois T.

Hasselt University, Belgium

Email: michelle.plusquin@uhasselt.be

Highly sensitive techniques for transciptome analysis, such as real-time PCR, microarrays and others currently used in functional genomics require a high integrity and quality of the RNA, as well as reproducibility between replicates of the same tissue. Our test-organism Macrostomum lignano is a small marine flatworm that has an average weight of 350 µg. Because culture of Macrostomum lignano is labour intensive our goal was to isolate RNA from small quantities of sample material. Samples were lysed with different mechanical lyses such as pulverisation, mixing with steal beads and sonication. Total RNA was then extracted using TRI- Reagent ®, as well as commercial kits based on RNA binding to silicon membranes (Qiagen, Roche) or magnetic beads (Invitrogen). RNA concentrations and purity was assessed using a nanodrop ® -ND 1000 UV-Vis Spectrophotometer using a 1 µl aliquot of the total RNA solutions. RNA integrity of the extracted RNA molecules was evaluated in 1 µl using an Agilent 2100 Bioanalyzer with the RNA 6000 Pico labChip® kit.

P 015

Automated RNA extraction using new Agencourt SPRI technology.

Souquet M.

Beckman Coulter, Germany

Email: msouquet@beckman.com

Agencourt RNAdvance is an Agencourt SPRI® paramagnetic bead-based purification system for the isolation and purification of total RNA from cultured eukaryotic cells, tissue or blood. Agencourt RNAdvance is a consistent and automation-friendly method for utilization in downstream microarray and real-time gene expression analysis. This technique reliably delivers high recovery and purity without the need for filtration or centrifugation and is especially well suited for automation using Beckman Coulter Biomek® solutions.

P 016

Advanced Data Mining Software for Applied Biosystems RT-PCR Data Analysis.

de Alarcon P.1 Ferlinz A.2

1Integromics SL, Spain and 2Applied Biosystems

Email: pedro.dealarcon@integromics.com

Great advances in instrumentation, accurate fluorescence detection, improvements in reagents (eg. by means of ABI Taqman probes) and protocols have increased the use as well as the range of applications of quantitative RT-PCR experiments. Nowadays, RT-PCR is the best technique for relative gene expression quantification and it is expanding to other areas like miRNA profiling, biomarker discovery, diagnostics and Copy Number Analysis. Moreover, the amount of data generated is also growing as experiments are being performed within a high-throughput context. In this sense, advanced bioinformatic tools that help researchers in the analysis and interpration of raw data are demanded by the scientific community as a key complement to the instrumentation and reagents. In the present poster, Integromics introduces a new high-performance software specifically designed for the new era of quantitative RT-PCR. The software has been built around four principles, namely: high-throughput statistics and data mining, interactive visualization, functional interpretation and extensible modular approach. State-of-the-art statistics are key to provide quality control and analysis of raw data for filtering of outliers and noisy experiments. Further procedures are provided towards the assesment of expression stability of endogenous genes (like Normfinder), differential expression by means of statistical tests, and clustering techniques for grouping similar expression profiles. All the statistics are implemented in the R language which ensures the capability of dealing with large amounts of data as well as benefiting from the existing libraries in Bioconductor (www.bioconductor.org). The software is implemented as a plugin of Spotfire Decisionsite, a leading application for professional and interactive visualization of multidimensional data. Visualizations in Spotfire facilitate the task of exploring large datasets (possibly with hundreds of genes and samples) so that it becomes easier to pose interactive queries and focus on relevant results. Even more, once the results from the analysis highlight the existence of characteristic expression profiles, it is crucial to enrich the numerical information with functional annotations provided by content repositories like the Panther database (www.pantherdb.org). As mentioned above, RT-PCR is a powerful technique that can be applied in many different contexts like relative quantification, miRNA profiling or genotyping. However, it is unrealistic to provide software that cannot be adapted to such diversity. In this sense, the software implements analytical workflows that can be easily adapted to specific user needs or experimental settings. A workflow is a set of sequential steps that guides the user from the importing of raw data to the final analysis in a very natural manner. This facilitates the use of the tool by non-experts in biostatistics but does not limit its power as statiticians can add or manipulate the procedures contained in the software.

P 017

Preamplification of Sample Limited Specimens for Real-Time Gene Expression Analysis.

Junko Stevens 1, Renata Coudry2, Cynthia Spittle3

1Applied Biosystems, California, US, 2Hospital A C Camargo,Sao Paulo, Brazil and 3Fox Chase Cancer Center, Philadelphia, US

Email: stevenjn@appliedbiosystems.com

Accurate gene expression profiling can be compromised by the quantity of RNA that is isolated from cells or tissues. We have developed a robust solution for uniform amplification of cDNA prior to quantitative, real-time PCR. TaqMan® Preamp Master Mix allows preamplification of up to 100 gene targets simultaneously using the TaqMan® Gene Expression Assays as the source of pooled gene-specific primers. TaqMan assay-based preamplification preserves equilibrium of targets and retains the relative copy numbers of starting targets in a reproducible and precise manner. Preamplification of random-primed cDNA is independent of amplicon distance from the 3’ end, making it amenable to use with partially degraded or viral RNA. Uniformity of preamplification was demonstrated using Laser Capture Micro dissection (LCM) in formalin-fixed, paraffin embedded (FFPE) tissue. RNA extracted from clinical samples was reverse transcribed to cDNA using High Capacity cDNA Reverse Transcription Kit The simple workflow enables researchers to enrich the amount of limited RNA samples uniformly within 1.5 hours.

P 018

qPCR with mRNA isolated from urothelial cells from urine.

Bontrup H., Delbanco J., Bruening T., Johnen G.

BGFA, Bochum, Germany

Email: bontrup@bgfa.ruhr-uni-bochum.de


Bladder cancer in chemical workers is an occupational disease associated with previous exposure to aromatic amines. Currently, urine-based markers used for screening of high-risk collectives are not of high sensitivity. To detect cancer at earlier stages more suitable non-invasive markers are necessary. Some promising new tumor markers are based on mRNA quantitation. The aim of this study was to establish and optimize a practical and efficient mRNA isolation method that allows applying qPCR-based assays with urothelial cells from urine.

Methods Six different isolation methods on the basis of commercially available kits were compared using urine samples of healthy donors and a control RNA with known concentration. A situation was simulated comparable to sample collection in a clinical setting. Cells were collected from urine by centrifugation and transferred to a buffer according to the manufactures recommendations. After short (48 h) storage at -20°C the mRNA isolation was performed. In all tested assays mechanical disruption of the cells was identical. The six isolation methods differed by DNA removal step (DNase treatment or special DNA column) and material of the RNA columns. After isolation, extracted RNA was transcribed to cDNA and quantified on a LightCycler system using an adapted Taqman-based assay for ß-Actin (FDI, Malvern, PA, USA). Possible DNA contamination was monitored with “RT-Minus-PCR” control runs.

Results It was possible to establish a system for mRNA isolation from urine but the analysis showed that urine as a starting material yields high contaminations with genomic DNA. Therefore, DNA removal is essential to obtain mRNA of sufficient quality for qPCR. Our results show that DNA could not be removed adequately by DNase digestion. Better yield and purity was only possible with application of a special glass fibre column that selectively binds DNA. This also allows to recover the DNA and use it for other applications.

Conclusions Most of the six tested methods for mRNA isolation from urine are generally suitable for downstream qPCR applications. However, best results can be obtained with a DNA column-based method (INVITEK, Berlin) that avoids DNase treatment. It excelled in two points: Reproducibility of yield even with very small amounts of starting material and reliability in the separation of DNA and mRNA. Both properties are an absolute requirement for field studies where cell material is limited and frequently of varying quality.

This study was in part supported by Fujirebio Diagnostics Inc.

P 019

Quantitative mRNA expression of the embryonic stem cell marker POU2 in early developmental stages of Atlantic cod (Gadus morhua).

Kaja Helvik Skjærven, Elisabeth Holen

National Institute of Nutrition and Seafood Research (NIFES), Norway

Email: ksk@nifes.no

Embryonic stem cells (ESC) from Atlantic cod (Gadhus morhua) have been isolated and cultured successfully. The cultured cells showed characteristic features for ESCs, including spontaneous differentiation and the ability to form embryoid bodies following retinoic acid treatment. In addition the ESCs could be directed to differentiate into neuronal like cells upon stimulation.

Detection of genes that identify ESCs from other differentiated cells is vital for the method. POU2 is one such gene, known to regulate potency, and self renewal in ESCs. Class V POU5f1 genes are found in blastula cells of different species; Oct4 in mammals and POU2 in zebrafish. We have previously described a fragment of POU2 isolated from Atlantic cod that is 100% identical to zebrafish POU2 at the amino acid level, but not at the nucleotide level. Using real-time PCR technique, we report that blastula cells taken from 1 and 1.5 days post fertilization (DPF) express the highest transcriptional levels of POU2. At the early gastrula stage (2 DPF) a 1.5 fold reduction in POU2 expression was correlated to the presence of some early differentiating cells. By the late gastrula stage (3 DPF), POU2 expression was barely detectable, and coincided with the presence of many differentiated cells. We conclude that POU2 can be used as an ESC marker for Atlantic cod, and the method represents an important new research tool for use in marine teleost species.

P 020

Relative mRNA quantification models and the impact of RNA integrity.

Simone Fleige and Michael W.Pfaffl

Physiology Weihenstephan, Center of Life and Food Sciences (ZIEL) Technical University of Munich, 85350 Freising, Germany

Email: fleige@wzw.tum.de

Quantitative real-time PCR has become one of the most widely used methods of gene quantitation. The method exhibits a large dynamic range, boasts tremendous sensitivity, has little to no post-amplification processing, and is amenable to increasing sample throughput. An essential requirement for a successful quantitative mRNA analytics using qRT-PCR is the usage of intact RNA. Low-quality RNA may compromise the derived expression results. The importance of RNA quality for the qRT-PCR was analyzed by determining the RNA quality of different bovine tissues and cell lines (n=11). Diverse artificial and standardized RNA degradation levels were assessed using the capillary electrophoresis system (Agilent Bioanalyzer 2100). The RNA quality was classified according the RNA integrity number (RIN). Further on, a research into the effect of different length of amplified products and RNA integrity on expression analyses was investigated. Degraded RNA interferes with qRT-PCR performance as such, expressed as Ct value, whereas PCR efficiency is minor effected by RNA integrity. Statements about importance of normalization could be confirmed by our investigations, consequently we commended an efficiency-corrected relative quantification strategy and normalization with at least one internal reference gene. We can recommend a RIN higher than five and a PCR product length up to 200 bp as a minimal requirement for a successful and reliable quantitative real-time RT-PCR quantification.

P 021


Pasi Kaarakainen1, Asko Vepsäläinen1, Aino Nevalainen1, Teija Meklin1,2

1National Public Health Institute, Department of Environmental Health, Kuopio, Finland and 2Technology Centre Teknia Ltd, Kuopio, Finland

Email: pasi.kaarakainen@ktl.fi

Possible specific associations between fungal species in our indoor environment and adverse health effects, such as respiratory symptoms, asthma and allergy are a major topic of interest. House dust sampling has been used to assess microbial exposures and to describe microbial populations in indoor environments. However, the repeatability or representativeness of such sampling has not been thoroughly validated.

In this study, the repeatability of house dust sampling was tested. Furthermore, the seasonal variation of the microbial populations was analyzed to assess the representativeness of a single sampling. QPCR was used for the identification of 16 species or assay groups of fungi from house dust samples from five houses without any moisture damage. The repeatability of the analysis of a floor dust sample with qPCR was tested by isolating DNA from homogenized dust samples as five parallel samples. Collecting samples in four different seasons enabled the consideration of seasonal variation.

The highest concentrations from analysed species or assay groups of fungi were observed for Aureobasidium pullulans, Penicillium/Aspergillus/Paecilomyces variotii group, Aspergillus penicillioides, Cladosporium herbarum and Cladosporium cladosporioides, respectively. Fungal concentrations varied between seasons depending on analysed species or assay groups. Concentrations of A. pullulans and two Cladosporium species were at their highest in summer and autumn while for Penicillium and Aspergillus species, the differences between different seasons were not so obvious. The repeatability of the parallel isolations of DNA was good for most of the analysed species. The best repeatability was observed for assays of C. herbarum and P. chrysogenum with ICC value (similarity of the parallel samples) 84.5 and 83.1 %, respectively. ICC was over 70 % also for six and over 60 % for three other assays. In summary, our results suggested that qPCR is a promising tool for the microbial analyses from house dust samples in epidemiological studies. In determining the qualitative procedures of the method and the reliability of the results, homogenization of the sample matrix and the number of the repeats of analyses are the primary issues.

P 022

RNA Integrity database: A web repository enabling RNA trace comparisons.

Hans Brunnert1, Martin Greiner1, Marcus Gasmann1, Michael Kim2, Marc Valer2

1Agilent Technologies, Waldbronn, Germany and 2Agilent Technologies Inc, Santa Clara, CA, USA

Email: martin_greiner@agilent.com

Lab-on-a-Chip devices are broadly used for RNA integrity analysis on Micro array and qPCR gene expression analysis. This creates the need and opportunity for users to screen and validate RNA traces for relevance and troubleshooting. Here we describe the design of the backbone as well as examples of use for a new RNA profile web database. The database aims to host a large variety of sample types spanning different genus, tissues and sample treatments, although the database is initially limited to contain bioanalyzer traces. Each electropherogram is annotated with sample source details as well as analytical data like UV, Ribosomal ratios, RIN and more. They also include details on the RNA extraction and the downstream experiment.

By design the database is open to the scientific community: free querying and curated contributions for individuals as well as large batch uploads from core labs. Individuals are able to compare their own results with those of others with similar samples and protocols. It also allows comparison of alternative RNA isolation methods based on its resulting electrophoretic traces.

Session:   qPCR BioStatistics & BioInformatics

Poster number   P 023    P 028

Location:           Student Cafeteria

P 023

GenEx for Real-time PCR Gene Expression Profiling.

Forootan A., Kubista M., Sjögreen B and Andrade J M

TATAA Biocenter, Sweden MultiD Analyses, Sweden Dept of Analytical Chemistry, University of A Coruna Center for Applied Scientific Computing, Lawrence Livermore National Laboratory

Email: amin.forootan@multid.se

The extraordinary sensitivity and virtually unlimited dynamic range of real-time PCR makes it the preferred technology for gene expression profiling. Candidate marker genes are identified by microarray technology and validated on representative samples by real-time PCR. False leads are discarded, resulting in very powerful panels of expression marker. Such panels can be developed for staging of disease, classification of cells, studies of expression pathways, effects of drugs and the like. The recent development of high throughput real-time PCR platforms such as the 384 well plate instruments from Applied Biosystems and Roche, and most recently the 48x48 (2304 wells) microfluidic card from Fluidigm will spur the development further. To extract maximum information from expression profiling experiments powerful statistical and mathematical methods are needed to find correlations between the measured profiles and identify the genes and samples that show common behavior. Here we present GenEx, which is the first windows based package dedicated for real-time PCR expression profiling. GenEx has user-friendly interface that makes advanced analyses really easy. Data from multiplate experiments are readily pre-processed and analyzed by methods such as geNorm , Normfinder, Principal Component Analysis, Hierarchical clustering, Self-Organizing Maps, Potential curves and much more. The results are presented in highly attractive plots.


The Real-Time Polymerase Chain Reaction, M. Kubista, J.M. Andrade, M. Bengtsson, A. Forootan, J. Jonak, K. Lind, R. Sindelka, R. Sjöback, B. Sjögreen, L. Strömbom, A. Ståhlberg, N. Zoric, Molecular Aspects of Medicine (2006) 27, 95-125

P 024

EM algorithm for gene copy number estimation using TaqMan® assays.

Catalin Barbacioru, Kelly Li, Raymond Samaha and Katherine Lazaruk

Applied Biosystems, Foster City, CA, United States of America

Email: catalin@appliedbiosystems.com

Genetic variation in the human genome takes many forms, ranging from large, microscopically visible chromosome anomalies to single-nucleotide changes. Deletions, insertions, duplications and complex multi-site variants, collectively termed copy number variations (CNVs) or copy number polymorphisms (CNPs), are found in all humans and other mammals. CNVs influence gene expression, phenotypic variation and adaptation by disrupting genes and altering gene dosage, causing diseases, as in microdeletion or microduplication disorders, or confer risk to complex disease traits such as HIV-1 infection and glomerulonephritis.

Recently, TaqMan® gene copy number assays have been developed for detection of genetic variation at gene level using primers and probes designed for genomic DNA sequences. Each well is duplexed with two assays. The FAM dye-based assay is designed to detect the genes-of-interest and the VIC® dye-based assay for detection of the reference gene, RNase P. The difference between FAM and VIC measurements (dCT) is indicative of the relative abundance of the gene-of-interest against 2 copies per diploid genome regardless of the status of the gene-of-interest. In this study, we present an algorithm for gene copy number estimation from TaqMan® gene copy number assays based on EM algorithm for mixtures of normal distributions. After removing technical outliers from data, the algorithm finds maximum likelihood estimates of parameters in probabilistic models, where the model depends on unobserved samples copy number of the gene-of-interest. Estimates of the gene copy number and confidence levels in predicted copy numbers are reported for each sample. Under current protocols, we are capable of distinguishing up to 8 copies of the gene of interest with at least 95% confidence, assuming 100% efficiency of the FAM dye-based assay.

To evaluate this algorithm, we present experimental results for 5 important drug metabolism genes (CYP2D6, CYP2E1, CYP2A6, GSTM1 and GSTT1) on 270 individual samples from International HAPMAP Project representing 4 different populations (Caucasian, African, Chinese and Japanese). Copy number analysis for these genes shows perfect consistency for sample duplicates. Copy number variation (from 0 to 4) is observed for all 5 genes. Significant differences of copy number frequency in these populations are revealed for CYP2A6, CYP2D6, GSTM1 and GSTT1. Therefore, data presented here is most relevant for highlighting variable regions of the genome that warrant consideration in disease studies. Furthermore, combining this data with SNP data for the same genes, we demonstrate that departures from diploidy can cause apparent genotyping failure and give inaccurate genotyping. Therefore, measuring copy number variation in these genes is an important complement to genotyping assays.

P 025

Statistical analysis of relative expression results in real-time PCR: Development of a Relative Expression Software tool (REST-2007©)

Ross Saad1, David Chiew1, Matthew Herrmann1, Valin Reja1, Michael W. Pfaffl2

1Corbett Research, Mortlake, NSW, Australia and 2Technical University Munich, Freising Weihenstephan, Deutschland

Email: michael.pfaffl@wzw.tum.de

REST 2007 is new standalone software for analyzing gene expression using real-time amplification data. The software addresses issues surrounding the measurement of uncertainty in expression ratios by introducing randomization and bootstrapping techniques. New confidence intervals for expression levels also allow measurement of not only the statistical significance of deviations but also their likely magnitude, even in the presence of outliers. Whisker box-plots provide a visual representation of variation for each gene, highlighting potential issues such as distribution skew. REST 2007 builds on its predecessor REST 2005 with significant improvements to randomization algorithms. This new revision introduces alternative data inputs such as single sample efficiency and amplification take-off point, alleviating the need to set amplification plot thresholds.


P 026

Using fuzzy logic algorithm and gene expression database ONCOMINE in COPD outcome forecasting.

Shilov B.V., Bukreeva E.B.

Siberian State Medical University (SSMU), Russian Federation

Email: shilov@ssmu.ru

Chronic obstructive pulmonary disease (COPD) occupies one of the first places in frame of a sickness rate in the world. COPD is a focus of chronic inflammation in organism. Chronic inflammation and its local repeated stress have long been known to be risk factors for cancer. Moreover risk of carcinogenesis increases under influence infectious pathogen. Examples include: Helicobacter pylor bacterial infection and gastric adenocarcinoma; hepatitis B virus and hepato-cellular carcinoma; chronic bowel disease and colon carcinoma; EBV and nasopharyngeal carcinoma in humans. Thus risk of malignant outcome of disease can be enlarged if a patient with COPD has colonization of the infectious agents in his organism.

Infectious agent of inflammation influences on squamous cell methaplasia development in COPD patients. This fact accompanies by increase of squamous cell quantity and rising in brush-biopsies reserved cell number belong in G2 phase of cell cycle. Our investigation included survey of 37 patients with COPD infectious exacerbation. In conditions of similar morphological and pathological changes different patients had various genes expression level.

There is known differences in these genes expression beside healthy humans and malignant disease patients. Dates about these differences were obtained from ONCOMINE database with free access. ONCOMINE, a cancer microarray database and web-based data-mining platform aimed at facilitating discovery from genome-wide expression analyses for elucidation state of indicated genes expression in patients with squamous cell methaplasia. Search and analysis ONCOMINE results we used in fuzzy logic algorithm for expert fuzzy rules forming.

When dealing with gene expression data, the problem is even more complicated, because no expert exists to determine what defines a “normal” expression level. Using fuzzy logic, the full range of expression data is first measured and is then broken into discrete subsections based on the observed data. These discrete subsections then provide a qualitative description of the data.

Use the algorithm has allowed patients classifying on the gene expression basis in groups with different forecast of the upshot of the disease. Three groups were chosen: favorable forecast (5 patients), stabile condition of the chronic inflammation (24 patients) and group with high probability squamous cell methaplasia development (8 patients).

P 027

qRT-PCR applied to the comparative analysis of plant splice variants in time-course water-stress experiments.

Cantale C., Latini A., Sperandei M. and Galeffi P.


Email: cantale@casaccia.enea.it

The understanding of the plant gene response to the environmental stresses at the transcriptional level is a main challenge for dissecting their role in the stress tolerance. We analysed the expression profile of a DREB-homologous gene, TdDRF1, using Real Time RT-PCR in various different Triticum durum and Triticale cultivars upon dehydration, both in greenhouse and in controlled field experiments. The TdDRF1 gene is alternatively spliced and results in three splice variants, two of them codifying for putative transcriptional activators and the third one producing an abortive putative protein with an unknown function. For each of the examined cultivars, a time-course experiment under water-stress is carried out including two groups, the control plants, that continue to be watered and the stressed plants, that are no more watered. The experiments are carried out using plants at the same growth level and some leaves are collected and pooled at fixed times to average the possible individual and position/soil variability.

The three splice variants, TdDRF1.1, TdDRF1.2 and TdDRF1.3 , are 1455bp, 1367bp and 1314bp respectively and a high sequence identity among the exons in the regions flanking the splicing recognition site prevents the design of primers able to select exclusively each transcript, as demonstrated by the co-amplification observed in experiments using SYBR Green technology. Thus, the qRT-PCR experiments have been carried out using the TaqMan probe-based technology and the 18S rRNA was used as an internal control. It has to be underlined that we were interested in comparing the three transcripts in the same samples of interest. As the expression levels of the three target genes are largely different and one is present in very small amounts, the experimental conditions resulted suboptimal in some cases, as demonstrated by the amplification plots.

Amplification efficiencies of each sample have been evaluated from raw data using LinReg software and show significant differences among the targets. The amplification efficiency of each transcript has been estimated also by linear regression using serial dilution. We report our results obtained using two different methods, Q-gene and a combination of LinReg software and GED formula. The last methodology has been applied using both individual and averaged amplification efficiencies. Furthermore, the application of the GED formula to our data results in three possible data combinations, that are compared and discussed.

P 028

Simplified simulation model of polymerase chain reaction (PCR).

Anneliese Ernst1, Veronique Creach2, Sven Becker3, Lucas J.Stal4, Peter M.J. Herman4.

1Fraunhofer IGB, Stuttgart, Germany, 2Cefas, Lowestoft, UK, 3Max Planck Institut für Limnologie, Plön, Germany and 4NIOO-KNAW, Yerseke, Niederlande

Email: ern@igb.fhg.de

A simplified model of polymerase chain reaction (PCR) is capable to describe essential characteristics of the amplification kinetics of this cyclic reaction. The model is based on the assumption that the PCR is designed and conducted in such a way that (one) PCR primer(s) limit(s) the formation of double-stranded PCR products. The absence of other limiting factors such as nucleotide limitation, enzyme limitation or shortage of the fluorescent dye SYBR Green® greatly reduces the number of parameters required to simulate real-time kinetics of individual amplification reactions in more explicit models. With a self-developed FORTRAN code, we can fit experimental data using the initial concentration of the limiting PCR primer to calibrate the fluorescence emitted by Sybr Green. This fit provides a novel method to estimate the initial concentration of target DNAs in a PCR sample. The method does not rely on external or internal standards. The simulations provide explanations for the occurrence of PCR biases and artifacts; and they are a valuable tool for teaching purposes, and in optimizing PCR protocols.

Session:  New Diagnostic applications with qPCR

Poster number   P 029    P 065

Location:           Student Cafeteria

P 029

Application of qRT-PCR in reproductive toxicology.

Wolfgang R. Schäfer, Wolfgang R. Deppert, Michael Simon, Katrin Roth, Aida Hanjalic-Beck, Claudia Nöthling, Hedwig Austermann-Hesse, Hans Peter Zahradnik

Universitäts-Frauenklinik Freiburg, Germany

Email: wolfgang.schaefer@uniklinik-freiburg.de

Drugs and xenobiotics are known to induce changes in gene expression which are often more sensitive and characteristic than currently employed toxicological endpoints. We describe the use of quantitative real-time RT-PCR in the development of an in vitro -test to assess possible hazards of chemicals on endometrial functions essential for embryo implantation. Our work is part of the integrated project ReProTect (EU) developing new -in vitro_-tests which are required under the new European chemicals legislation (REACH) to reduce, refine or replace the use of laboratory animals. Within the ReProTect research area of “Implantation” we are exploring on the mRNA level predictive toxicological endpoints in human endometrial explants (e.g. leukaemia inhibitory factor, LIF; progesterone receptor; estrogen receptor α; calcitonin; cyclooxygenase-2; corticotrophin releasing hormone receptor 1; VEGF-receptor 2, KDR).

The goal of the human endometrial explants assay is to detect chemical effects on endometrial functions which are essential for embryo implantation. This test is a closer approach to in vivo -conditions than primary or permanent cell cultures. Endometrial tissues from the proliferative and secretory phase of the menstrual cycle are obtained by aspiration curettage from premenopausal women. The endometrial biopsies are chopped into pieces of 1-2 mm/side and cultured in sex steroid supplemented medium without phenol red in a humidified atmosphere at 37 °C in 5 % CO<sub>2 for 6-24 hrs. Test chemicals are administered during this incubation period and compared to negative controls with vehicle alone.

Facing the low tissue amounts of human endometrial explants which are available for each incubation (approximately 20 mg) quantitative real-time RT-PCR (LightCycler 480, Roche) is applied as the major analytical method. In order to identify predictive toxicological endpoints among a larger number of target genes assays from the highly versatile Universal Probe Library (UPL; Roche) are used. Evaluation is performed by calibrator-normalized relative quantification. A status report of our project will be presented. It includes calibrator standard curves for efficiency correction, selection of housekeeping genes and preliminary data of in vitro -testing with the antiprogestin reference compound RU 486.

This project is funded by the European Commission ( ReProTect , Project no.: LSHB-CT-2004-503257)

P 030

Borrelia burgdorferi sensu lato complex in Ixodes ricinus ticks in Slovenia and development of SYBR Green I melting curve analysis for separation between different borrelia genospecies.

Nataša Toplak1, Miha Kovač2, Minka Kovač1, Tjaša Cerar3 and Eva Ružić Sabljić3

1Omega d.o.o., Dolinškova 8, 1000 Ljubljana, 2Poljane Grammar School Gimnazija Poljane, Strossmayerjeva 1, 1000 Ljubljana and 3University of Ljubljana, Faculty of Medicine, Institute of Microbiology and Immunology, Zaloška 4, 1000 Ljubljana

Email: omega@omega.si

Worldwide, many infections caused by viruses, bacteria, or parasites are known to be tick-transmitted zoonoses. Tick-borne diseases are a major threat to mammals and humans during outdoor activities in spring and fall. One of the most important tick-borne diseases is Lyme borreliosis caused by spirochetes within the Borrelia burgdorferi sensu lato complex. The different species of this group are involved in clinical manifestation of disease. The purpose of this study was to investigate the prevalence of Borrelia burgdorferi sensu lato in ticks collected from different regions in Slovenia during summer of 2005. The ticks were collected by the cloth-dragging method. Ticks were mechanically crushed and semi-automated nucleic acid extractions were performed. All DNA samples were tested by real-time TaqMan PCR for successful DNA isolation and to confirm the presence of B. burgdorferi sensu lato . To differentiate between the B. burgdorferi sensu lato genospecies, B. burgdorferi sensu stricto , B.garinii and B. afzelii different approaches of real-time PCR with SYBR Green I melting curve analysis of different part of borrelia genome was used.

P 031

Composition analysis of mesophilic mixed dairy starters by quantitative PCR.

Brockmann E., Schlichter K.

Chr. Hansen A/S, Denmark

Email: elke.brockmann@dk.chr-hansen.com

Mesophilic dairy starters typically consist of Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis biovar. diacetilactis and Leuconostoc species. Traditionally the total count and the percentages of Leuconostoc spp. and of L. lactis subsp. lactis biovar. diacetilactis have been determined by plate counting. Leuconostoc and L. lactis subsp. lactis biovar. diacetilactis percentages are of importance in the various production processes of dairy products manufactured with the help of mesophilic starters. Plate counting is laborious and often requires long incubation times before a result is available. Therefore we developed quantitative PCR assays that allow to specifically quantify the different components of undefined mixed dairy starter cultures. Most of the assays were targeted to ribosomal RNA genes. Where assay design was difficult because of closely related sequences, LNA technology was successfully applied. The poster will present aspects of assay design and performance for the composition analysis of mesophilic dairy starter cultures by quantitative PCR.

P 032

Copy Number Variation Analysis on the HapMap DNA Samples Using TaqMan® Assays for CYP2D6, CYP2E1, CYP2A6, GSTM1 and GSTT1.

Kelly Li1, Catalin Barbacioru1, Fiona Hyland1, Kashif A. Haque2, Robert A. Welch2, Caifu Chen1, and Katherine Lazaruk1

1R&D, Applied Biosystems, Foster City, CA, US and 2Division of Cancer Epidemiology and Genetics, SAIC Frederick, National Cancer Institute, Gaithersburg, MD, US

Email: chencx@appliedbiosystems.com

Gene copy number variation is now recognized as an important type of polymorphism in the human genome. Gene duplication or deletion can have a significant impact on phenotype. Gene copy number polymorphisms have been associated with genetic diseases such as cancer, immunological and neurological disorders, and have also been associated with variations in drug response. For example, copy number changes for drug metabolism genes such as GSTM1, GSTT1, and CYP2D6 are known to be associated with variations in response to various drugs. Accurate detection of copy number changes is critical for understanding how gene copy number variation plays a role in disease or drug response. We have developed real-time quantitative PCR assays to quantify copy number using TaqMan® technology for a number of genes, including 5 important drug metabolism genes: CYP2D6, CYP2E1, CYP2A6, GSTM1 and GSTT1. The method involves relative quantification of the gene of interest versus a reference gene known to be 2 copies per diploid genome. Relative quantity is determined by the delta-delta Ct method, where the endogenous control is RNase P, and the calibrator is a DNA sample known to have 2 copies of the gene of interest. The TaqMan-based duplex gene dosage assay is an accurate, robust, and reproducible method for gene copy number detection. Here we report the copy number results for the CYP2D6, CYP2E1, CYP2A6, GSTM1 and GSTT1 genes using DNA samples from the International HapMap Project. The HapMap DNA samples come from a total of 270 individuals representing the populations with African, Asian, and European ancestry. There are 30 sets of trio samples (two parents and one child) from Yoruba people of Ibadan, Nigeria; another 30 sets of trios from U.S. residents with northern and western European ancestry; 45 unrelated individuals from Japan and 45 unrelated individuals from China. The gene copy number data has been useful in understanding the SNP genotype data from the HapMap samples. For example, most of the GSTT1 HapMap SNPs failed QC (were qc-), due to either a pass rate <80%, a low Hardy-Weinberg p-value, or to Mendelian inconsistencies. All of these inconsistencies within the SNP genotyping data can be explained when the gene dosage data are factored into the analysis.

P 033

Detailed investigation of the retroviral life cycle using real-time PCR assays.

Steinrigl A., Ertl R., Klein D.

Vetomics Technologiezentrum der VUW, Austria

Email: Dieter.Klein@vu-wien.ac.at

The transduction efficiency of viral vectors can easily be monitored during pre-clinical trials by inclusion of marker genes (Klein et al. (1997) Gene Therapy 4, 1256-1260). However, the use of such marker genes has to be avoided in the final clinical gene therapy situation since their products often represent powerful immunogens and lead to the elimination of transduced cells. Thus it is not desirable to use such genes as markers in clinical settings, especially if the vector is applied in vivo. In these cases PCR based methods like the real-time PCR might provide a powerful tool to estimate biodistribution (Klein (2002) Trends in Molecular Medicine 8, 257-260). In order to investigate the accuracy and precision of this method, we have developed and tested a real-time PCR assay for the quantification of the enhanced green fluorescent protein (EGFP) gene and compared the results with transduction efficiencies estimated by FACS analysis (Klein et al. (2000) Gene Therapy 7, 458-463). Another important topic of retroviral vectorology is to minimize the risk of insertional tumorgenesis. In order to avoid this potential risk, targeted integration into the host genome would increase the safety of retroviral vectors and might provide a first step towards gene repair. The first step towards this long-term goal is the inhibition of the retroviral integration machinery without any impairment of earlier steps in the retroviral life cycle. Therefore, we constructed several MLV integrase mutants defective in either the catalytic centre of IN, the C-terminal region, or both. Viral particle production, RT-activity and infection efficiency of the IN mutant vectors were tested in comparison to a vector harbouring wild type integrase in single-round transduction assays. Furthermore, the kinetics of reverse transcription and nuclear entry were analysed using a set of quantitative real-time PCR assays. While none of the introduced mutations seemed to substantially influence particle production, moderate reductions in RT-activity could be observed. Although all mutants were integration defective, integration was not completely abolished, which is in accordance with the results of other groups. Surprisingly, however, only one mutant, containing an almost complete deletion of the C-terminal domain, was impaired in the accumulation of late reverse transcription products, while a mutant with a similar deletion encompassing the complete C-terminus plus part of the catalytic domain showed normal kinetics of reverse transcription. Nuclear entry was not influenced by any IN mutation or deletion. These results corroborate the work of others stating that MLV IN, independent from its catalytic activity, also has an influence on reverse transcription and suggest that the molecular basis for this function might reside in several discrete parts of the protein.

P 034


Kärkkäinen P., Valkonen M., Nevalainen A. and Rintala H.

National Public Health Institute, Department of Environmental Health, Laboratory of Environmental Microbiology, P.O.Box 95, 70701 Kuopio, Finland

Email: paivi.karkkainen@ktl.fi


Aim of the research is to develop a QPCR-application for determination of Gram-positive and Gram-negative bacteria from environmental samples.


Primers and dual-labeled TaqMan-probes were designed for 16S rRNA- gene location based on sequences retrieved from NCBI Gene Bank. In order to find possible areas for specific primers and probes, multiple sequences from 20 Gram-positive and 19 Gram-negative bacterial families were aligned first separately and then together using the Vector NTI AlignX software (InforMax Inc., Bethesda, MD,USA). One set of primers and two probes were designed so that both probes matched within the same 144 bp amplification product. Formation of secondary structures and primer dimers was checked with the NetPrimer software (PREMIER Biosoft International, Palo Alto, CA, USA). Primer and probe concentrations and reaction temperature were optimized. The optimized reactions included 2x concentrated Dynamo Probe QPCR-mastermix (Finnzymes Oy, Espoo, Finland), 400 nM of primers, 100 nM of probes and 50 ng/µl of template DNA in a reaction volume of 25µl. All reactions were pipetted with Corbett CAS-1200 pipetting robot and run with Corbett Rotor-Gene 3000 using the 0.1 ml PCR tubes (Corbett Life Science, Sydney, Australia). The function and specificity of the QPCR -assay was tested with separate and duplex probes and different bacterial and fungal strains.


Sensitivity of the assay was not equal for both probes, due to competition between the probes since they bind in same gene location in 16S rRNA. Reaction efficiency was slightly better for the Gram-negative probe than the Gram-positive (100 % vs. 93 %).

Specificity and reaction efficiency of the application was tested with Bacillus cereus, Staphylococcus aureus, Streptomyces californicus, Mycobacterium mucogenicum, Escherichia coli, Enterobacter aerogenes and Pseudomonas aeruginosa , respectively. Some of the Gram-positive bacteria tested, Streptomyces californicus and Mycobacterium mucogenicum , remained undetected due to a one base pair difference in the middle of the probe, in contrast all three Gram-negative bacteria were determined successfully. All 15 different fungal strains gave negative results.

The Ct values for non-template control reactions in the Gram-negative assay were consistently between 26,38 and 29,45, whereas in the Gram-positive assay they remained undetected. This is most probably due to background coming from the mastermix, since the recombinant DNA-polymerase is produced in E. coli . The background could be avoided by using individual reagents and a native polymerase instead of a commercial mastermix.

P 035

Tumor suppressor gene RBSP3 in cervical carcinoma: copy number and transcriptional level.

Anedchenko E. A. 1, Kisseljova N. P. 2, Dmitriev A. A. 1, Kisseljov F. L. 2. and Zabarovsky E. R. 3, Senchenko V. N. 1

1Engelhardt Institute of Molecular Biology, Moscow, Russian Federation, 2Blokhin Cancer Research Center, Moscow, Russian Federation and 3MTC, Karolinska Institute, Stockholm, Sweden

Email: anedchenko@rambler.ru

Chromosomal aberrations and expressions deficiencies in genes at the 3p21.3 region are frequently found in most human cancers including cervical cancer. RBSP3 (RB1 serine phosphatase from human chromosome 3, other symbols CTDSPL , HYA22 ) is a tumor-suppressor gene from AP20 (3p22-p21.33) region. The protein activates pRb by dephosphorylation and participates in negative regulation of cell cycle. The aim of this study was comparative analysis of RBSP3 copy number and transcriptional level in HPV-positive squamous cervical carcinomas (SCC) of I-III stages.

Copy number and mRNA level quantification were done on 76 DNA and 59 cDNA samples from tumor and normal tissues using TaqMan technology and comparative or ∆∆Сt method (ABI PRISM 7000 SDS, Applied Biosystems). All validation experiments were performed previously. It was shown the good reproducibility of data in the same and different runs, low variability of reference genes β-actin and GAPDH in normal and tumor tissues. We used different comparators (paired adjacent norm, average norm from several patients and DNA from lymphocytes) for calculations of copy number and transcriptional level of RBSP3 gene. We calculated the efficiency of all reaction for the target and references in tumor and norm. For this purpose we developed the program “Analysis of Expression Genes In paired Samples” (AEGIS) program (Reg.No. 006613816, RF).

Deletions were detected in 42% of cases (19 of 45 studied biopsies). The frequency of deletions was significantly higher in SCC samples with metastases (64%) than in tumors without metastases (32%, P<0.05). In a few cases amplification of RBSP3 was also found. Altogether copy number changes of RBSP3 were detected in 51% of cases (23 of 45). The expression of RBSP3 was decreased in 64% of SCC samples (21 of 33). Again decreased expression of RBSP3 was detected significantly more frequently (83%) in tumors with metastases compared with SCC without metastases (52%, P<0.05). In several cases however increased expression was observed. Altogether changes in expression of RBSP3 were detected in 79% (26 of 33) of SCC biopsies. Comparative analysis showed that in 23% of SCC cases decreased expression of RBSP3 was detected in samples with deletions and in 36% cases such decrease was not associated with copy number changes. Rarely more complicated SCC cases were found. For example in some tumors increased expression of RBSP3 was detected in samples with deletions or without changes of copy number. Results of the study suggested that RBSP3 is involved in the progression of SCC, and there is more than one mechanism of its inactivation. Obtaned data can be useful for diagnosis of SCC.

This work was supported by the INTAS 03-51-4983 and Russian Foundation for Basic Research Grant 05-04-49408 .

P 036


Francesca Salvianti1, Pamela Pinzani1, Raffaella Grifoni2, Costanza Paoletti2, Alessandro Ciani3, Francesca Ucci3, Cinzia Mazzini3, Bruno Neri2, Mario Pazzagli1

1Department of Clinical Physiopathology, University of Florence, Italy, 2Department of Oncology – Centre of Experimental and Clinical Oncology, University of Florence, Italy and 3Department of Otoneuroophtalmology, Eye Clinic,University of Florence, Italy

Email: m.pazzagli@dfc.unifi.it

In recent years, researchers’ interest has been focusing on investigating the importance of circulating tumor cells (CTC) in solid cancers as a prognostic marker, even if the biological significance of this phenomenon is still under debate. The Real Time PCR approach to detect CTCs consists in designing a set of primers and probe recognizing a target sequence which is tumor or tissue specific.

The most common molecular marker used for CTCs detection in cutaneous melanoma is tyrosinase mRNA, which is expressed only by melanocytes and melanoma cells. In literature many papers deal with cutaneous melanoma, while uveal melanoma has been by far less investigated. A high expression of melanoma-associated RNA (tyrosinase, MART1 and gp100) has been described in patients with liver metastases from uveal melanoma.

The objective of our work is to develop a method to accurately quantitate tyrosinase mRNA in blood and evaluate the potential of this parameter as a prognostic marker in uveal melanoma. In particular, we are interested in investigating the predictive value of tyrosinase mRNA levels in case of relapse. Our purpose is also to evaluate the reliability of this marker in monitoring the effectiveness of therapies.

Twenty patients with uveal melanoma (47-79 years old) were involved in this study, after receiving their informed consensus, and followed longitudinally for a period of about one year. As a negative control, 20 subjects undergoing surgery for detached retina were enrolled. Total RNA was isolated from whole blood by the PAXgene technology (PreAnalytiX). Primers and Taqman flourogenic probe for tyrosinase were purchased from Applied Biosystems. To calculate the expression of tyrosinase mRNA in each sample, we referred to an external reference curve generated with Quantitative PCR Human Reference Total RNA (Stratagene).

The method adopted for tyrosinase mRNA quantification was validated by means of calibration curves. These standard curves showed linearity over the entire quantification range (250 ng – 25 pg total RNA). A typical standard curve equation was: y = -3.32 x + 40.47. The correlation coefficient was 0.99.

According to our preliminary data, tyrosinase mRNA appears as a good candidate to become a prognostic marker in uveal melanoma.

A significant relationship was found between tumour size and tyrosinase mRNA levels. Moreover an association between the marker and response to chemotherapy was evidenced, opening a perspective for its usage in monitoring therapy efficacy. Finally, patients with a primary involvement of the ciliary body showed a significantly higher expression of the marker than controls. This can be explained by the presence in this tissue of many blood vessels and consequently a direct release of circulating tumour cells.

Further studies are required to elucidate the potential of such a molecular marker, as follow up has been too short to assess anything about overall surviving and disease recurrence.

P 037

Development of a highly sensitive quantitative detection system for viable cells of Listeria monocytogenes.

Petersen A.

Danisco Deutschland GmbH, Germany

Email: antje.petersen@danisco.com

Listeria monocytogenes is an opportunistic bacterial pathogen that accounts for significant human food borne diseases worldwide. The high risk of listeriosis for especially pregnant women and immunocomprised people require technologies that allow the sensitive identification and quantification of L. monocytogenes in situ. Conventional microbiological methods for detecting L. monocytogenes in food products are time-consuming. Therefore, faster DNA based quantitative polymerase chain reaction (qPCR) and mRNA based quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays were developed in the present study.

An analysis of different Listeria strains showed that the chosen primers and probes were specific for the detection of L. monocytogenes by qPCR.

Minced meat and hotdog samples were seeded with L. monocytogenes in the range of about 101-108 cells per gram sample. Samples were then processed for bacterial concentration using high-speed centrifugation followed by DNA or RNA extraction (Stevens and Jaykus, 2004).

The application of the most sensitive DNA-based assay developed in the present study achieved quantifications as low as 1 colony-forming unit per PCR reaction. The availability of a rapid molecular assay being able to quantify 1 CFU per PCR reaction is of great value for assessing the risks of food borne listeriosis.

One disadvantage of DNA-based methods is that they do not distinguish between living and dead cells. This was confirmed by spiking raw meat with DNA of L. monocytogenes that was still detectable after 72 h at 4°C. Among the different types of nucleic acids messenger RNA (mRNA) is the most promising candidate as an indicator of viability in bacteria. In addition, we could show in the present study that mRNA spiked into raw meat was completely degraded after 4 h at 4°C. Because of the central importance of transcription for catabolism and anabolism synthesis of mRNA is a meaningful indicator of viability.

Therefore, a quantitative reverse transcription PCR (qRT-PCR) strategy was established to quantify short-living mRNA of L. monocytogenes in food matrices. Application of the qRT-PCR assay to quantify L. monocytogenes cells in artificially contaminated meat products achieved sensitivities of 19 CFU/PCR reaction. Positive amplification results for prs mRNA were achieved for all replicates when 7.4 x 104 CFU/g minced meat or 2 x 103 CFU/g hotdog where tested in the qRT-PCR giving a linear response over 5 orders of magnitude. Using qRT-PCR, the number of detectable cells of L. monocytogenes was about 1750 CFU/g.

In conclusion, qPCR and qRT-PCR are effective and precise technologies for the quantification of L. monocytogenes cells in food matrices. The developed qRT-PCR assay has already been implemented in Danisco’s food safety development labs.

P 038

Real-time multiplex RT-PCR on circulating tumor cells.

Anieta M. Sieuwerts, Stefan Sleijfer, Jaco Kraan, Joan Bolt-de Vries, Jan-Willem Gratama, John WM Martens and John A. Foekens.

Department of Medical Oncology, Erasmus MC, Rotterdam, The Netherlands

Email: a.sieuwerts@erasmusmc.nl


Metastases are thought to develop from CTCs, tumor cells circulating in the peripheral blood. The presence of > 5 CTCs in 7.5 ml whole blood was found to predict clinical outcome in metastatic breast cancer (Cristofanilli et al., NJEM, 2004). Gene expression profiling of CTCs is likely to improve outcome prediction in breast cancer as this may yield better insight into mechanisms underlying dissemination and drug sensitivity. Since one human cell contains 5-10 pg total RNA and traditional real-time RT-PCR requires 5-20 ng RNA, a pre-amplification step is required to analyze expression of multiple genes. This study aimed to establish a method to perform mRNA expression analysis on as little as 5 CTCs in 7.5 ml whole blood.

Materials & methods

Two to 20 human breast cancer cells were spiked in 7.5 ml whole blood of healthy donors. Using the CTC kit (CellSearch™), cells that attached to anti-EpCAM Moab were immunomagnetically separated and used for analysis of a selected pilot set of 32 genes by real time RT-PCR. Three linear pre-amplification methods were compared (Pre-amp gene-specific amplification (Early Access, ABI); Whole Transcriptome Ovation RNA amplification (NuGEN); and Full spectrum RNA amplification (System Bioscience)). Unsupervised two-dimensional hierarchical cluster analysis was performed for comparing mRNA profiles of human breast cancer cells with the mRNA profiles of healthy blood donors and blood spiked with human breast cancer cells after EpCAM enrichment.


Of the methods tested, the Pre-amp method from ABI was superior in terms of homogeneous amplification of small amounts of mRNA. With this method all tumor-specific genes were reproducibly quantitated in RNA isolated from only one tumor cell. Since qRT-PCR data from pre-amplified RNA from 5 tumor cells clustered with non-amplified RNA from 1000 tumor cells, pre-amplification did not compromise gene expression profiling. However, gene expression analysis of EpCAM purified cells isolated from blood of healthy donors showed that some contaminating blood cells in the EpCAM-purified CTC-fraction exhibited mRNA expression of EpCAM and other genes thought to be tumor-specific. The presence of EpCAM or non-specific EpCAM binding to Fc receptors may account for this contamination. Nevertheless, already several tumor-specific genes (e.g., MUC-1 and SPDEF) could be detected in samples containing only 2 tumor cells spiked in 7.5 ml whole blood.

Discussion & conclusion

This study shows the feasibility of multiple gene expression analysis on RNA isolated from only one tumor cell. Furthermore, pre-amplification does not compromise gene-expression profiling. Most importantly, expression analysis of several tumor-specific genes in blood samples containing only 2 tumor cells is already possible. To further optimize CTC characterisation, additional purification steps are required to reduce the leukocyte contamination in EpCAM purified CTC fractions.

P 039

Development of a quantitative TaqMan LNA (“Locked Nucleic Acid”) qPCR assay for determining viral load of HCV-infected patients.

Moreira E.S. and Alberto F.L.

Fleury Institute, Brazil

Email: flalberto@gmail.com

We developed a real-time quantitative RT-PCR assay for determining the viral load of HCV infected individuals employing TaqMan LNA probe. Tests were designed to be prone to automation from RNA extraction from primary PPT tubes. 400 uL of plasma were submitted to RNA extraction on the Qiagen 9604 Biorobot and the isolated RNA was re-suspended in 100 ul of Qiagen elution buffer. 20 ul was added to each qPCR reaction to a final volume of 50 ul with “Taqman EZ RT-PCR" kit (Applied Biosystems). The amplified segment comprises 102 bp of the 5’untranslated region of the HCV genome. The probe, which includes 5 LNA nucleotides, was designed with the help of the softwares “Primer Express” (Applied Biosystems), "LNA probe optimizer" (http://lnatools.com/hybridization/) and "Exiqon Tm prediction" (http://lna-tm.com/). All oligonucleotides were checked for conservation within “HCV Sequence Database" (http://hcv.lanl.gov/content/hcv-db/BASIC_BLAST/basic_blast.html). The standard curve was produced with an amplified segment of 270 bp of the same HCV region in vitro transcribed, purified and serial-diluted in yeast tRNA 100 ng/uL. OptiQuant™ HCV RNA Panel (Acrometrix) was used as an external control, which allowed the conversion of final results from copies to IU/mL. Each sample was also submitted to BCR gene transcript amplification as the extraction control. All the assays were performed in the RotorGene 3000 (Corbett Research). The test detected all the HCV subtypes available (1a, 1b, 2a, 2b. 2c, 3a, 5) with linearity from 400 to 108 IU/mL. HCV RNA was inconsistently detected in dilutions containing less than 50 UI/mL, which was the 2 SD limit of detection (95% CI). As compared to other widely used methods the higher throughput and linearity range as well as the reduction of the contamination risk represent a step forward in meeting the requirements of the routine clinical laboratory.

P 040

Development of a real-time PCR assay for quantitative analysis of human influenza A virus replication.

Vester D. 1, Seitz C. 2, Bettenbrock K. 2, Genzel Y. 2, Reichl U. 1,2

1Otto-von-Guericke-University Magdeburg, Bioprocess Engineering, Magdeburg, Germany and 2Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Magdeburg, Germany

Email: vester@mpi-magdeburg.mpg.de

Influenza viruses cause yearly epidemics with considerable impact on health and economics. In pandemic years hundreds of millions of cases occurred throughout the world with a high rate of mortality. To design effective antiviral drugs or vaccines for preventing influenza pandemics a detailed understanding of intracellular processes of host cells during an influenza virus infection is essential. The general course of events during influenza virus replication in host cells are well understood, but much about dynamics and control of expression of the viral genome as well as the transcription of the viral messenger RNAs still remains unclear.

In this work, we apply real-time PCR to investigate the time course of human influenza A virus replication in a transformed MDCK (Madin Darby Canine Kidney) cell line. We designed a two-step technique based on a well-established and frequently used method described for influenza virus detection. In the first step, total cellular RNA was extracted from influenza virus infected MDCK cells or from cell culture supernatant during infection. In a second step complementary DNA was synthesized in a reverse transcription using polarity specific primer: producing complementary DNA of either viral RNA of negative polarity (vRNA(-)) or of complementary RNA and messenger RNA of positive polarity (cRNA(+), vmRNA(+)) for influenza virus. Finally, these transcripts were analysed in a quantitative real-time PCR using SYBR green fluorescence.

Here, we present first results in designing and optimising this sensitive and specific two-step quantitative real-time PCR technique.

The main focus of future investigations will be a comparative analysis with simulation results of a structured mathematical model of influenza virus replication in MDCK cells. It is expected that experimental results clarify basic assumption on the control of viral protein synthesis and viral genome replication.

P 041

Differential expression of 27 apoptosis related genes in vitamin d-induced differentiation of HL-60 cells.


Kocaeli University, Turkey

Email: aylinkanli@yahoo.com

Using a quantitative real- time PCR (LightCycler), we analyzed 27 genes ( bcl2, bcl-xl, bcl-w, mcl1, bik, bax, aif, survivin, caspase 3, caspase 6, caspase 7, caspase 9, cytochrome C, fas, myc, TGF beta, ERK1, JNK1, p38 mapk, p15, p21, p27, CDK2, CDK4, Cyclin D1, Cyclin D2, Cyclin E) for changes in expression associated with the apoptosis of human promyelocytic leukemia HL-60 cells induced by 1alpha,25-dihydroxyvitamin D3 at various time points 18, 48 and 72h. Cells were cultured and RNA samples were isolated. Relative quantification data obtained from PCR products of cDNA portions. We did not find distinct down or up-regulated expression profiles at these time points. Findings suggest that there are not clear apoptotic signals in early phases of differentiation and the genes involved in vitamin D-induced apoptosis of HL-60 cells would be more clearly visible after the terminal differentiation process. Apoptosis and cell cycle analysis could be performed in this experimental setting, using quantitative real- time PCR.

P 042


Vincenzo Calvanese, Aitor Sánchez-Fernández (*), Fernando Carrasco-Ramiro (*) and Begoña Aguado

Centro de Biología Molecular Severo Ochoa (CBMSO), Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain. (*) Servicio de Genómica CBMSO

Email: fcarrasco@cbm.uam.es

Alternative splicing is described as one of the most important ways to generate proteome complexity, starting from a relatively limited number of genes. Among the five main classes of alternative splicing, intron retention is probably the less understood.

Detailed transcriptome studies of the Major Histocompatibility Complex (MHC) class III region genes indicate a high rate of different splicing events. This genomic region has a particular biomedical interest due to the immune-related diseases mapped in this area, which cannot be fully ascribed to a particular gene. There are evidences of disrupted regulation of splicing processes in pathology and, to study this phenomenon, we need analytical tools not only to identify, but also to precisely quantify, transcripts which often differ in only few nucleotides.

We have transcriptionally analysed the MHC class III region LY6G5B gene, belonging to the LY6 superfamily, mainly composed of cysteine-rich extracellular proteins involved in cellular interaction. Studying the LY6G5B expression in different cell lines, tissues and organisms, by classic RT-PCR, we found that there is an abundant transcript retaining the first intron of 148 nucleotides. This transcript cannot be translated to a protein, due to a premature stop codon, and it should be degraded quickly by the Non-sense Mediated Decay (NMD) machinery. Nevertheless, it seems to be stable and even the most abundant transcript, especially in tissue samples. This could indicate that this mis-spliced form is a real transcript which could have a potential regulatory function.

This work focuses on the design of a real-time PCR assay to distinguish expression levels of the correctly spliced and the intron retained form. We optimised the primer design and the reaction conditions to avoid the cross-reactivity of the two transcripts. We also excluded the interference of genomic contamination working in parallel on RT-minus samples, as the intron-retaining transcript does not differ at all from the genomic sequence. We generated standard dilution curves, using the two isoforms cloned in a plasmid, to achieve an absolute quantification, in order to compare their absolute quantity in the same sample. Currently, with the final assay set-up, we are measuring the stability of the two transcripts in different cell lines to ascertain differences on expression levels, to later correlate them with potential disease associations.

In conclusion we optimised a real-time-PCR experiment designed for the differential detection of two really similar templates.

P 043

Discover the Differences – Gene Expression Analysis with Universal ProbeLibrary: Now available as Dual-Color Assays, and for New Organisms.

R. Mauritz, R. Rein, R. Beck, C. Goldstein

Roche Diagnostics, Roche Applied Science, Germany

Email: Ralf.Mauritz@Roche.com

Although quantitative real-time RT-PCR is in principle a simple technique, the assay design remains fairly complex, and assays designed for specific applications often lack sensitivity and reproducibility. The time spent on assay design, optimization, and validation often becomes a bottleneck in the implementation of new assays for large-scale expression profiling.

The Universal ProbeLibrary is a fast, specific and flexible format for quantitative real-time PCR experiments. The concept is based on the fact that just 90 short probes modified with locked nucleic acids (LNA) provide transcriptome-wide coverage in most organisms (human, mouse, rat, arabidopsis, drosophila, primates and C. elegans ). Universal ProbeLibrary probes can be designed by using a free, web-based assay design software, called ProbeFinder. In seconds, ProbeFinder designs highly specific intron-spanning assays for a target transcript. The system allows multiple design options, including designs for transcript variants and gene family members.

The results presented here demonstrate that the Universal ProbeLibrary is now available for relative quantification experiments by multiplexing with housekeeping gene assays suitable for many real-time instruments. Additionally, new features implemented in the assay design software ProbeFinder conveniently integrate the positions of known SNPs into the assay design process, and visualize the SNP positions on the screen. Finally, we extended the Universal ProbeLibrary assays to new organisms ( e.g. , maize, rice, zebrafish) delivering the same performance, and success rates observed in already existing organisms.

P 044

Universal ProbeLibrary – Evaluation of its Applicability Regarding qPCR Instruments.

E. Fernholz, D. Heisswolf, R. Mauritz, R. Rein, O. Seth

Roche Diagnostics, Roche Applied Science, Germany

Email: Erhard.Fernholz@Roche.com

The Universal ProbeLibrary is a fast, specific and flexible format for quantitative real-time PCR experiments. The concept is based on the fact that just 90 probes provide transcriptome-wide coverage in most organisms (human, mouse, rat, arabidopsis, drosophila, primates and C. elegans ). The probes are short using the LNA technology to reach appropriate melting temperatures (Tm). Since each probe is short and can therefore bind to several locations along the DNA, the specificity of each assay depends on the interplay of selected primers and the Universal Probe. However, each assay is designed with a convenient, free of charge web-based assay design software, called ProbeFinder. The system facilitates multiple design options, including designs for transcript variants and gene family members.

Different PCR-programs may influence the assays. Each qPCR-instrument has its own unique features (e.g., ramp rates). Here, we evaluated the performance with different UPL-assays on several real-time PCR instruments.

P 045

Downregulation of huntingtin affects expression levels of interaction partners and morphological changes in neuronal and HeLa cells.

Manuel Ammerschläger, Sandra Ritz, Kerstin Bieser, Andreas Holloschi, Mathias Hafner and Petra Kioschis

University of Applied Sciences, Institute of Molecular and Cell Biology, Mannheim, Germany

Email: m.ammerschlaeger@hs-mannheim.de

Huntington's Disease (HD) is a neurodegenerative disorder caused by an abnormally expanded polyglutamine tail in the amino-terminal region of huntingtin (htt). Pathogenic mechanisms involve a gained toxicity of mutant htt and a potentially reduced neuroprotective function of the wild-type allele. Among the molecular abnormalities reported, HD cells are characterized by the presence of aggregates, transcriptional deregulation, altered mitochondrial membrane potential and disturbed calcium (Ca2+) signalling. The biological function of htt has not been completely elucidated. It is reported that short interfering RNA (siRNA)-mediated inhibition of endogenous htt results in an aberrant configuration of the endoplasmic reticulum (ER) network in vitro in different cell lines. We aimed to investigate htt down-regulation mediated effects on the ER in different human neuronal cell lines combined with gene expression profiling of 20 different htt interaction partners. Our results indicate that the ER in different neuronal cell lines tested and the expression level of several htt interaction partners are sensitive to htt down-regulation. Only weak htt expression levels in Ntera2 cells were detected on basal expression profiles and some interaction partners like PSD95 were not or weakly expressed in non-neuronal HeLa cell lines.

P 046

Expression of candidate ivermectin resistance genes in Sarcoptes scabiei.

Mounsey K. 1, Holt D. 1, McCarthy J. 2, Currie B. 1,3 and Walton S. 1

1Menzies School of Health Research, Institute of Advanced Studies, Charles Darwin University, Darwin, Australia, 2Queensland Institute of Medical Research and Australian Centre for International and Tropical Health and Nutrition, University of Queensland, Brisbane, Australia and 3Northern Territory Clinical School, Flinders University, Darwin, NT, Australia

Email: kate.mounsey@menzies.edu.au

Scabies is an infectious skin disease caused by the burrowing ectoparasitic mite Sarcoptes scabiei . Ivermectin is currently the treatment of choice for hyperinfested (crusted) scabies, and has been identified as a potentially effective acaricide for mass treatment programs in scabies endemic communities worldwide. Recent reports of ivermectin resistance in scabies mites raise concerns for the sustainability of such programs. It is therefore critical to define the molecular mechanisms of ivermectin resistance to enable early detection of emerging resistance.

The objective of this work was to investigate the expression levels of candidate ivermectin resistance genes from S. scabiei . These included a novel chloride channel gene and representative members of the glutathione S-transferase and ABC transporter gene families.

Mites obtained from crusted scabies patients were separated according to life stage and ivermectin exposure. qRT-PCR was performed using SYBR green for eight target genes, amplified in parallel with B-actin, which allowed for normalisation of varying cDNA concentrations and assessment of inter-assay reproducibility. Data were analysed using the relative quantification methods of Pfaffl.

Glutathione S-transferase genes were highly expressed at all developmental stages of S. scabiei . Expression of the chloride channel and ABC transporter genes were comparatively low. Of particular interest, a multidrug resistance protein and glutathione S-transferase were up-regulated in ivermectin exposed mites by 6 and 2.5-fold respectively compared to non exposed mites. This increase in expression was most pronounced in adult female mites. The potential involvement of these proteins highlights a previously unexplored mechanism of ivermectin resistance.

To our knowledge, this is the first application of qRT-PCR to S. scabiei . Molecular studies on this organism have been traditionally limited due to insufficient genetic material. These approaches are giving us new insights into scabies mite biology and the basis of emerging ivermectin resistance. This may eventually assist in overcoming many of the current difficulties in monitoring treatment efficacy and allow more sensitive methods for monitoring emerging resistance in the community.

P 047

Fast real-time PCR for the detection of tanapox virus and yaba-like disease virus.

Pia Zimmermann 1, Ingo Thordsen 2, Hermann Meyer (1*)

(1) Bundeswehr Institute for Microbiology, Neuherberger Str. 11, 80927 Munich, Germany (2) Eppendorf Vertrieb Deutschland GmbH, Peter-Henlein-Str. 2, 50389 Wesseling-Berzdorf, Germany (*) Corresponding author: hermann1meyer@bundeswehr.org

Email: thordsen.i@eppendorf.de

Agents like tanapox virus (TPV) and yaba-like disease virus (YLDV) may infect humans leading to symptoms which can be mistaken with smallpox, monkeypox, tularemia and anthrax, caused by pathogens possibly used for deliberate bioterroristic offenses. To ensure the most efficient cure for infected patients and the cooperation of public health organisations in case of emergency, a reliable and rapid detection of the causative agent is of outstanding interest. We report the development of the first ultra fast real-time PCR assay for the detection of TPV and YLDV using real-time PCR instruments of the latest generation. Mastercycler® ep realplex4 S (Eppendorf, Germany) and Mx3000P® (Stratagene®, Germany) were used to optimize an established TaqMan® probe based assay for the detection of both, TPV and YLDV. The proposition of this approach was to reduce the running time as short as possible without loosing more than one logarithmic dilution step sensitivity. We were able to reduce total running time from 2:14 hours from the original published thermo profile down to 48 minutes (Mx3000P®) and 22 minutes (Mastercycler® ep realplex4 S), respectively. Probit regression analysis demonstrated, under comparable experimental conditions, a minor sensitivity loss of the assay on the Mx3000P® platform and of about one logarithmic dilution step on the Mastercycler® ep realplex4 S platform.

P 048

A Novel Multiplex, Quantitative Gene Expression Approach for Breast Cancer Biomarker Research.

Souquet M.

Beckman Coulter, Germany

Email: msouquet@beckman.com

Quantitative gene expression analysis is playing an increasingly important role in cancer research. Currently available techniques utilize microarray analysis to detect the expression of a large number of genes per reaction, or alternatively, utilize real-time PCR to detect the expression of a few genes (at lower throughput). A multiplex approach to analyze a specific set of genes from a given biological pathway in a single reaction using a limited amount of RNA is of great interest to cancer biologists. To address this demand, the GenomeLab™ GeXP Genetic Analysis System was developed. This system utilizes eXpress Profiling technology, a highly multiplexed PCR approach to quickly and efficiently analyze the expression of 20-30 genes with high sensitivity. The throughput is scalable to over 6000 gene expression results per day. Here we present a study using a GeXP-designed marker panel of 29 genes whose functions are directly or indirectly related to breast cancer formation. Our study demonstrates that this approach can quantitatively detect the expression of the genes in this multiplex cancer panel in a single reaction using as little as 25 ng of total RNA isolated from human breast cancer tissues. The GeXP system can also be used to design custom multiplex panels to evaluate biological pathways involved in other forms of cancer. The GenomeLab GeXP Genetic Analysis System is a cost-effective way of performing multiplex gene expression analysis with scalable throughput capacity, high assay robustness, and excellent data quality.

P 049

Gene expression changes of energy metabolism regulators in adipose tissue of women with gestational diabetes mellitus.

Petra Kleiblova1, 2, Zdenek Kleibl1, Vratislav Krejci2, Ivana Ticha1, Martin Haluzik3

1Institute of Biochemistry and Experimental Oncolgy, 1st Faculty of Medicine, Charles University in Prague, Czech Repbulic, 2Dept. of Obstetrics and Gynecology 1st Faculty of Medicine, Charles University in Prague and General Faculty Hospital, Czech Republic and 33rd Dept. of Medicine 1st Faculty of Medicine, Charles University in Prague and General Faculty Hospital, Czech Republic

Email: pekleje@lf1.cuni.cz

Gestational diabetes mellitus (GDM) is the condition of glucose intolerance appearing in woman usually in the second trimester of pregnancy with prevalence 5-7% of pregnancies worldwide. GDM is considered to be an independent risk factor for subsequent type 2 diabetes mellitus development. The aim of our study was to characterize expression profile of genes participating to the energy metabolism regulations in adipose tissue pre-selected by gene expression array.

METHODS: The women with single pregnancy parturient by Caesarean section were enrolled into the study. All subjects gave the informed consent approved by local ethical committee. Subcutaneous and visceral (omental) adipose tissue samples were obtained during surgery from each of analyzed subjects: 15 women with GDM (9 treated by s.c. insulin and 6 treated by a diet regimes) and 11 healthy slim women in control group with physiological pregnancy. Mean age and gestational age at the day of delivery were comparable in all groups. The tissue samples were preserved in RNAlater (Qiagen) in –20°C until total RNA isolation by RNeasy Lipid Tissue Mini Kit (Qiagen) according to manufacturer. Genes with differential gene expression were identified using gene expression array targeted to 128 genes involving to insulin receptor pathway (Human Insulin Signaling Pathway Microarray; Superarray) in a group of 5 patients with GDM (3 treated by insulin, 2 by diet regimes) and 3 healthy controls. Expressions of selected genes were analyzed by qPCR using SybrGreen (LightCycler 2.0; Roche) and quantified by REST-384-beta software with normalization to 3 house-keepings (B2M, GAPDH and PBGD).

RESULTS: The most apparent changes in visceral adipose tissue of pregnant women with GDM involved up-regulation of NOS2A (NO-synthase 2A) mRNA (2.4x; p=0.0125) and down-regulation of PRL (prolactin) (11.5x; p=0.0165) and IGFBP1 (IGF-binding protein 1) (59x; p=0.003) comparing to healthy cohort. Moreover, in subgroup of obese pregnant women with GDM (BMI>30 kg.m-2 at the 3rd day after the delivery) the pronounced decrease of expression of PRL (25x; p=0.008) and IGFBP1 (194x; p=0.003), together with decreased expression of IRS2 (insulin receptor substrate 2) (1.5x; p=0.0425) was found (comparing to healthy controls).

In subcutaneous adipose tissue of GDM patients the two-fold increase of LDLR (LDL receptor) (p=0,0065) was found. In a sub-group of pregnant women with GDM treated by insulin the down-regulation of IRS2 (1.7x; p=0.044) was detected.

CONCLUSIONS: The current results indicate 1) substantial alteration of gene expression of insulin pathway regulators in GDM patients; 2) differential involvement of subcutaneous/visceral adipose tissues on the pathogenesis of GDM. Currently running prospective study will score the ability of gene expression profiling for the early diagnostics of the type 2 DM risk development in GDM patients.

ACKNOWLEDGMENT: Supported by IGA MZCR 8302-5 and Research project MSM 0021620814.

P 050

Host responsiveness during low dose infection with different strains of BCG.

Tania Di Pietrantonio 1, 2, Manon Girard 1, Annie Verville 1, Marianna O. Orlova 1, Adam Belley 1, Marcel A Behr 1,3 and Erwin Schurr 1,2,3.

1Centre for the Study of Host Resistance, McGill University, Montreal, Quebec, Canada, H3G 1A4, 2Department of Human Genetics, McGill University, Montreal, Quebec, Canada, H3A 1B1 and 3Department of Medicine, McGill University, Montreal, Quebec, Canada, H3G 1Y6

Email: tania.dipietrantonio@mail.mcgill.ca

RATIONALE: The anti-tuberculosis vaccine Mycobacterium bovis Bacille Calmette Guérin (BCG), is a widely administered vaccine despite its variable protective efficacy. Since BCG substrains differ in their production of antigenic proteins, it is possible that strain-specific components are involved in the quality of host response mechanisms. Here, we investigated host responsiveness to three strains of BCG in two mouse strains with contrasting innate susceptibilities to BCG.

EXPERIMENTAL APPROACH: A/J and C57BL/6J mice were infected intravenously at 8 to 10 weeks of age with approximately 104 colony forming units (CFU) of BCG Russia, Japan or Pasteur. Candidate gene transcriptome analysis of the lung and spleen using real-time PCR was performed at 7, 21, and 42 days post-infection. Bacterial enumeration was obtained in parallel.

RESULTS: Among the three BCG substrains tested, BCG Pasteur stimulated the largest production of interferon gamma (Ifng). BCG Pasteur, which grew progressively better in the spleens of the C57BL/6J strain at day 21, also preferentially induced higher levels of both Ifng and interleukin-12b (Il12b) in this mouse strain. Although continuous growth was observed for BCG Russia in the lung and spleens of both host strains, induction of Ifng and Il12b was observed in C57BL/6J animals only. Infection with BCG Japan, which was characterized by markedly low splenic bacterial load and an absence of pulmonary bacterial growth, failed to evoke an immune response in both strains of mice. Differences in the production of interleukin-4 (Il4) across BCG strains were not observed.

CONCLUSION: Employing quantitative PCR, we determined that genetically heterogeneous strains of BCG elicit different host immune responses. Using comparative genome analysis of BCG strains, it may be possible to correlate these distinct host response traits with specific bacterial genome elements.

P 051

Investigating molecular diagnosis of tuberculosis using transrenal DNA. The nucleic acid extraction step.

Lorenz A1, Hoelscher M1, Gerhardt M2, Minja F2, Morris-Jones S3, Zumla A3, Huggett J3

1Department of Tropical Medicine and Infectious Diseases, Ludwig Maximilian University, Munich, Germany, 2Mbeya Medical Research Programme, Mbeya P.O. Box 2410, Tanzania and 3Centre for Infectious Diseases and International Health, University College London, UK

Email: Andreas.Lorenz@campus.lmu.de

Tuberculosis (TB) kills roughly four people every minute and represents a major global health problem. Despite this fact diagnosis relies on antiquate methods like sputum stain microscopy, culture or empirical assessment. With the exception of culture (which typically takes six weeks), the efficacy of the available diagnostic methods are both poor and highly variable. Nucleic Acid Amplification Techniques have not broken this trend despite the existence of a number of commercially available methods. A major reason for this is the lack of comprehensive optimisation when “in house” methods have been used. Optimisation steps have not always been applied to all the stages required to store, process and assess a clinical specimen. The most common specimen, when diagnosing TB, is sputum, which can be highly heterogeneous and difficult for nucleic acid extraction. We are interested in the use of urine for molecular diagnosis of TB. Urine represents the ideal clinical sample as it is easy to collect, with minimal risk of infection. There are a number of studies describing the detection of Mycobacterium tuberculosis (Mtb) DNA from TB patients using PCR. However they suffer from the same optimisation criticisms mentioned above. The nucleic acid extraction methods where not tested for the recovery of DNA from urine or the removal of inhibitors that may disrupt the PCR reaction. Furthermore none of these studies consider that the target DNA is likely to be part of the Circulating Nucleic Acids in Plasma and Serum (CNAPS). From which molecules of ≤150 bp are reported to cross the kidney barrier and to be found in urine as transrenal DNA (trDNA). Consequently for efficient detection of these molecules PCR reactions with amplicons of <150 bp are desirable. We have developed an Mtb complex specific qPCR assay which will be assessed for diagnostic utility. However before such an assessment can be made the pre-PCR steps have to be optimised. The work described here aims to establish an efficient method for extracting trDNA from urine. We have spiked urine from a number of healthy male donors with different sized Mtb DNA sequences. By re-extracting these DNA molecules using a number of different DNA extraction methods we aim to investigate their respective efficacies for both DNA recovery and the removal of inhibitors. Identification of an efficient trDNA extraction procedure will enable us to accurately assess the utility of targeting Mtb trDNA as a diagnostic method for TB.

P 052

Monitoring of Resistance Genes during Antibiotic Therapy of Pigs.

Hölzel C., Bauer J.

TU München, Lehrstuhl für Tierhygiene, Germany

Email: christina.hoelzel@wzw.tum.de

Tetracyclines are the most common antibiotics in livestock farming. The correlation between application of antibiotics and spread of antibiotic resistance is beyond question, but hard to quantify. Thus a quantification method for tet(M) and tet(O) in pig liquid manure was established. In a first approach, we tried to follow the rise of tetracycline resistance genes in faeces during oral application of chlortetracycline (CTC) to pigs.

Material and Methods

For the evaluation, liquid manure from antibiotic free raised pigs was treated by x-rays (up to 409 kGy) in a 60Co-radiation-facility in order to destroy bacterial resistance genes. This pre-treated manure was artificially contaminated with different concentrations of Bacillus “R89” which posseses exactly one copy of tet(M)/cell. DNA extraction was carried out with PowerSoilTM DNA-Isolation Kit. The content of tet(M) was analysed by means of real-time PCR (Light Cycler®, Roche; detection mode: SYBR®GreenI). Recovery rates were determined by comparing the number of copies analysed versus the number of copies added.

The course of the number of tet(O) and tet(M) in the faeces during the application of CTC (20 mg/kg body weight) was studied in two different trials (A/B). In trial A, samples of faeces of 6 antibiotic free reared pigs were examined for tet(O) before, during and after the application of CTC. In trial B, 2 groups of pigs (n=5) were fed a CTC-containing and a CTC-free diet, respectively. Each pig was kept in a metabolic cage within the same stable. Faeces were analysed daily for the content of tet(M)-copies using qPCR.


Exposure to x-rays reduced the tet(M) content in the manure by one order of magnitude per 54 kGy. The analysis of the artificially contaminated samples (103 to 107 copies per capillary) showed recovery rates from 15.8 to 26.7 % with a standard deviation between 0.4 and 14.3 %.

The medication of pigs with CTC (trial A) resulted in a significant increase of tet(O) copies in faeces from 6.92 log10/g at the beginning to a maximum of 8.45 log10/g at day 6 of the withdrawal period. The decrease of the copies during the withdrawal period was very slow; at the end a concentration of 7.66 log10/g was still measured.

In trial B, the numbers of tet(M) copies in the faeces of the medicated pigs lay nearly one order of magnitude higher than those of the control (8.31 vs. 7.46 log10/g). However, the course of the mean values of the control group was nearly parallel to that of the treated group, indicating a transfer of resistant bacteria via air and dust.

Future prospects

Monitoring of resistance genes during and after application of distinct antibiotics enables to assess the particular selection potency of the antimicrobial agent. Thus, it might offer interesting information for decisions of choice between equally effective antibiotics as well as for decisions of market authorization.

P 053

Preliminary evaluation of the GeneXpert Dx System for CML patients monitoring through the Xpert BCR-ABL MonitorTM assay: comparison with traditional RT-qPCR methods.

Silvia Calatroni, Barbara Rocca, Ilaria Giardini, Marina Boni, Irene Dambruoso, Paolo Tarantino, Paolo Bernasconi

Division of Hematology - IRCCS Policlinico S. Matteo Foundation, Pavia, Italy

Email: s.calatroni@smatteo.pv.it

The molecular signature of BCR-ABL fusion gene in chronic myeloid leukemia (CML) provides a unique tool for diagnosis and monitoring of tumor burden during therapy. The introduction of imatinib mesylate, allowing the achievement of high rates of clinical and cytogenetic remission, has revolutionized the treatment of CML patients and reinforced the fundamental role of BCR-ABL transcript levels monitoring by RT-qPCR to assess minimal residual disease. Nevertheless, many procedural aspects of this complex technique require a strong inter-laboratory optimisation and recommendations for harmonizing the different methodologies have recently been proposed. The Xpert BCR-ABL MonitorTM, recently developed by Cepheid (Sunnyvale, CA 94089-1189), is an In Vitro Diagnostic assay, whose intended use is the molecular monitoring of p210 BCR-ABL transcript in peripheral blood samples of CML patients, through a fully automated platform, the GeneXpert Dx System (Cepheid). The instrument can perform nucleic acid isolation, reverse transcription and qPCR into a multi-chamber single-use disposable cartridge thanks to the integration of a quantitative real-time thermal-cycler with a software-driven cartridge processor. The analysis, that requires only 200 microlitres of sample, is completed in less than 2 hour. We will illustrate and discuss data collected from 20 CML samples (19 peripheral blood and 1 bone marrow sample), corresponding to 19 patients submitted to imatinib therapy, analysed with the Xpert BCR-ABL MonitorTM assay. Results of the comparison with both a home-made SybrGreen-based RT-qPCR method and a commercial Taqman-based In Vitro Diagnostic assay (M-bcr FusionQuant® Kit, Ipsogen, France) will be also presented.

P 054


Ion Gutierrez-Aguirre1, Andrej Steyer2, Mateja Poljšak-Prijatelj2, Jana Boben1, Kristina Gruden1 and Maja Ravnikar1

1Department of Plant Physiology and Biotechnology, National Institute of Biology, Ljubljana, Slovenia and 2Institute for microbiology and immunology, Faculty of medicine, University of Ljubljana, Ljubljana, Slovenia

Email: ion.gutierrez@nib.si

Rotaviruses are the major cause of acute diarrhea in animals, infants and children under 5 years old. They are unenveloped viral particles with three proteinaceous layers and their genome consists of 11 dsRNA segments. Six structural proteins (VP1–VP4, VP6 and VP7) and six non-structural proteins (NSP1–NSP6) are encoded in the viral genome. Rotaviruses are classified into 7 groups (A–G) based on the antigen specificity of the VP6 gene. The most common is group A, infecting both humans and animals. According to the VP4 and VP7 antigenic and molecular characterization, group A rotaviruses are further classified into different p and g types. The most frequent genotype combinations in humans are G1 P[8], G2 P[4], G3 P[8], G4 P[8] and G9 P[8]. These are also the most predominant global genotypes.

In case of natural disasters, i.e., earthquakes, floods, etc., fecal waters can mix with potable waters. Rotaviruses can then, along with other pathogenic agents, be present in water supplies constituting a possible risk for the population. The development of a fast and sensitive qPCR detection method which could detect very low rotavirus concentrations and cover as many rotavirus genotypes as possible would help to prevent such risks.

We have designed a qPCR TaqMan approach targeted to the VP2 gene, based on a multi-sequence alignment of 7 different human rotaviral strains. To overcome the high nucleotide sequence diversity within the different rotaviral strains, multiple forward and reverse primers were designed in addition to a MGB TaqMan probe. The approach was tested on seven infected stool samples, each containing one of the following genotypes: G1 P[8], G2 P[4], G3 P[8], G4 P[8], G8 P[8], G9 P[8], and G12 P[8]. A sample containing a mixture of all seven genotypes was also tested. Initial concentration of samples was assessed by electron microscope counting using latex beads of known size and concentration. Three different RNA isolation procedures were tested: TRIzol, thermal denaturation and the magnet bead based King Fisher method. RT-qPCR was performed in two separate steps. Preliminary results show that we are able to detect rotavirus genotypes that significantly differ at the sequence level. Limit of detection for the method used is acceptable with ≥90% efficiency values. This approach will be used in future attempts to detect the presence of rotaviruses in water samples.

P 055

Quantification of live bacteria by quantitative PCR.

Bennedsen M., Koivalo S.

Chr. Hansen A/S, Denmark

Email: mads.bennedsen@dk.chr-hansen.com

Quantitative PCR is a powerful tool for studying population dynamics of bacteria in complex samples, e.g. the population dynamics of starter cultures in cheese ripening. The major drawback of the use of PCR based methods is, that it measures genomes regardless of the viability of the bacteria. Unfortunately DNA of killed bacteria is stable for weeks in biological samples, compromizing the quality of the qPCR based data. A method for distinguishing live and dead Gram negative bacteria has previously been published (Rudi et al 2004)

In the present work we describe methods for enumeration of live Gram positive bacteria, using one of two reagents: Ethidium monoazide bromide (EMA) and propidium monoazide iodide (PMA).

EMA treatment of live and dead Lactobacillus sp show a reproducible shift of 10 Ct of the dead bacteria as compared to the live bacteria, corresponding to a reduction of the signal from dead bacteria by 99,9%.

P 056


Olatz Villate1, Aitor Sánchez-Fernández 2, Fernando Carrasco-Ramiro 2 and Begoña Aguado1

1Centro de Biología Molecular Severo Ochoa (CBMSO), Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid, Madrid, Spain and 2Servicio de Genómica CBMSO

Email: oli_bio@hotmail.com

Alternative mRNA splicing is a complex post-transcriptional mechanism. It enables the generation of different mRNA isoforms from the same primary transcript, allowing a large diversity of proteins to be synthesised from a relatively small number of genes. There is a growing interest in precisely quantify splice variants expression. In addition, alteration of normal splicing events can cause or modify human disease. For this, a reliable method for measuring the expression levels of splice variants is an important step in investigating the significance of each isoform. Quantitative real-time PCR is the most sensitive method to ascertain gene expression levels that has been developed to date.

Our aim is to applicate real-time PCR technology to quantify the different splice variants generated by genes, located in the Major Histocompatibility Complex class III region, which are potentially implicated in diseases. We are now focusing in the NFKBIL1 ( nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-like 1 ) gene, which is a susceptibility gene for developing reumatoid arthritis. It encodes a protein resembling members of the IkappaB protein family that regulate bioavailability of NFkappaB. We have identified two splice variants of this gene, which differ on the 5´UTR.

Here we describe the design of a real-time PCR assay that differentiates these two variants using a TaqMan probe and the use of a method which creates reliable standard curves using plasmids containing the alternative transcripts. To start, we have set up this method in cell lines and have observed that the representative transcript is the mainly expressed in Raji cells (78%) and in Hek293 cells (86%), corresponding to human B and embrionary kidney cells, respectively.

As this NFKBIL1 gene can be involved in the development of reumatoid arthritis, we now want to quantify whether the ratio of the splicing isoforms might change among patients and/or correlates with disease severity. We are beginning to analyse mRNA extracted from blood and synovial fluid samples of patients with reumatoid arthritis and blood mRNAs from controls. We want to applicate this technology to the study of another genes potentially implicated in diseases.

P 057

Quantitative PCR based assessment of colon colonisation by 4 lactobacilli in an orally consumed synbiotic formula.

Nadal A.1; Aldeguer X.2; Coll A.1; Gonzàlez-Huix F.2; Acero D.2 and Pla M.1

(1) Institut de Tecnologia Agroalimentària, Universitat de Girona, Spain (2) Servei de Cirurgia General i Aparell Digestiu, Hospital Universitari Josep Trueta, Girona, Spain

Email: anna.coll@udg.es

Probiotics and prebiotics have been reported to have beneficial effects on human health in various clinical situations; and to act through diverse mechanisms such as direct effects in the intestinal lumen, or indirect effects by modulation of the endogenous microflora or of the immune system (Marteau et al., 2004). Four different lactic acid bacteria (LAB) strains obtained from ecologic cultivated rye and oats were chosen for use in a synbiotic formula ( Synbiotic 2000 ) due to their strong bioactivity and resistance to gastric and bile acids (Kruszewska et al., 2002). Recent results show that this synbiotic formula leads to significant clinical improvement to certain types of patients (Pathmakanthan et al., 2002); and further prospective assays are currently taking place (financed by the Fundació LaMarató de TV3).

Aiming at the quantitative traceability of the four LAB in Synbiotic 2000 , real-time PCR (QPCR) assays were designed and optimised for the specific detection and quantification of each LAB in colon biopsies. Sequences from luxS , putative pheromone pcrA , kinase acetate and collagen adhesion 1158 genes were selected as QPCR targets. They all fit the following criteria i.e. (i) display percentages of homology below 70% among the target LAB species and other sequences in gene databases; and (ii) exhibit complete homology to all sequences from different strains of the same species or subspecies.

A total of 78 bacterial (mainly LAB) stranis were assayed and the four QPCR assays proved fully specific and highly sensitive, with detection limits of 10 target molecules per reaction with 95% probability. The assay was linear along 6 orders of magnitude (R2 > 0.99) and accurate quantification was possible down to a limit of around 30 target molecules. Comparison of the results obtained by QPCR and standard microbiological methods showed an excellent relative accuracy. The assay was further adapted to the analysis of colon biopsies by incorporation of a pre-PCR treatment; and proved capable to accurately quantify down to 100 target LAB cells per biopsie with detection limit of 10 cells (30 % probability).

We used the developed QPCR assays to show the colon colonisation by the 4 LABs. Additionaly we concluded that the developed QPCR assays were a valuable tool for on-going studies of probiotics colonization dynamics.

Kruszewska D, Lan JG, Lorca G et al. Selection of lactic acid bacteria as probiotic strains by in vitro tests. Microecology and Therapy 2002; 29: 37-51.

Marteau P, Seksik P, Lepage P and Dore J. Cellular and physiological effects of probiotics and prebiotics Mini Rev Med Chem. 2004; 4(8):889-96.

Pathmakanthan S, Walsh M, Bengmark S et al. Efficacy and Tolerability treating acute distal ulcerative colitis with Synbiotic enemas: A Pilot Trial. Gut 2002; 51:A307.

P 058

Rapid and sensitive detection of infectious poxvirus particles.

Andreas Nitsche, Daniel Stern and Georg Pauli

Robert Koch-Institut, Germany

Email: nitschea@rki.de

Since the anthrax spore attacks occurred in fall 2001 in the USA, the potential abuse of variola virus or genetically engineered orthopoxviruses for bioterrorist plots has been intensely discussed. To date, several diagnostic assays have been reported that allow the rapid and reliable detection of poxvirus particles or poxvirus genomes in suspected samples. Electron microscopy (EM) can rapidly prove the presence of poxvirus particles. However, EM cannot differentiate between orthopoxvirus species and is limited in sensitivity, since reliable detection is only possible with particle concentrations >106/mL. Molecular methods like real-time PCR are much more sensitive, detecting less than 10 genome equivalents per PCR reaction, but PCR can only prove the presence of short stretches of poxvirus DNA. Nevertheless, both EM and PCR fail when an evaluation of the infectious capability of the viral particles is required because they can not discriminate between infectious and non-infectious virus particles or nucleic acids. In clinical samples taken from patients presenting with typical symptoms of a poxvirus infection, it is usually sufficient to prove the presence of viral particles by EM to diagnose an infection whereas detection of poxvirus nucleic acids permits the differentiation of variola virus infection from infection with other orthopoxviruses. In both cases, the clinical symptoms are usually attributed to a virus which is considered to be replication competent and infectious. In contrast, in environmental samples including suspect parcels, it is essential to prove that a virus-positive result indicates infectious particles in order to allow a reasonable risk assessment. The fastest diagnostic approach that allows the identification of infectious poxvirus particles is propagation in a suitable cell culture system and detection of poxvirus proteins using specific antibodies which takes at least one day. Therefore, we have combined the cell culture approach that can doubtlessly demonstrate virus replication, with the speed and sensitivity of real-time PCR. To increase sensitivity, we changed the target of real-time PCR from poxvirus DNA to poxvirus mRNA genes which are highly expressed during the first few hours of the infection cycle. Expression levels of these genes enable sensitive detection 1 to 2 hours after inoculation. The complete diagnostic approach can prove the presence of as little as 3 PFU of infectious poxvirus particles in less than 5 hours. This extremely low detection limit indicates that environmental samples, which may contain cell culture inhibitory substances, can be diluted by several orders of magnitude in order to dilute inhibitors while maintaining the viral load at detectable levels.

P 059

Real-time PCR in diagnostics of plant viruses.

Mehle N., Boben J., Ravnikar M.

National Institute of Biology, Slovenia

Email: natasa.mehle@nib.si

For identification of plant viruses ELISA, immuno-serological electron microscopy, test plants and RT-PCR or PCR are frequently in use. Recently, a more sensitive and specific method real-time PCR was developed for diagnostics of certain plant viruses. Real-time PCR can be used for determination of different viruses as well as for differentiation between related strains of viruses. The method can also be used for detection of low concentrations of plant viruses in various samples such as environmental waters, growth substrates, and seeds.

In our laboratory real-time PCR was developed for identification of Chrysanthemum stem necrosis virus (CSNV). The method for detection of serologically closely related Tospovirus Tomato spotted wilt virus (TSWV) (Boonham et al., 2002) was successfully implemented in the diagnostic procedures. Previously we demonstrated that CSNV serologically cross-reacts with many commercially available antisera against TSWV in ELISA assay and that the symptoms on chrysanthemum are very similar in both, CSNV and TSWV (Ravnikar et al., 2004). Real-time PCR used alone or in combination with other methods is a way for efficient and reliable detection and differentiation of this two Tospoviruses.

Concentration of plant viruses in irrigation waters and soil is usually below the sensitivity of frequently used detection methods such as ELISA. Real-time PCR for identification of Tomato mosaic virus (ToMV) in tomato and tobacco plants, irrigation waters and soil was developed. The method was shown to be 1000 times more sensitive than ELISA (Boben et al., 2006). Real time PCR was also shown to be more sensitive than other methods in detection of Potato virus Y (PVY) in potato leaves (Mehle et al., 2004).

In real-time PCR reactions used for detection of viral presence in the plant material the internal controls 18S or COX (Boonham, personal communication) were included in the assays in order to control the RNA extraction procedure. Reverse transcription step was performed separately or one-step real-time RT-PCR was performed and compared with two-step assay.

P 060


Alfred Schöller1,3, Christa Freibauer1,3, Eva-Maria Dlouhy-Schütz2,3, Min Wang4, Youji Hu4, Mark Stearns4, Gerhard Lunglmayr3

1Institute of Clinical Pathology, LK Weinviertel Mistelbach, Austria, 2Department of Urology, LK Weinviertel Mistelbach, Austria, 3Karl Landsteiner Institute for Andrology and Prostate Research, LK Weinviertel Mistelbach, Austria and 4Department of Pathology and Laboratory Medicine, Drexel University, Philadelphia, USA

Email: alfred.schoeller@mistelbach.lknoe.at

Objectives: In the last ten years hundreds of genes which are over-/under-expressed or specifically produced in prostate cancer tissue have been discovered. The growing prostate tumor exfoliates cells (molecules) into spontaneous urine via the urethra during natural shedding/apoptosis/lysis. In addition the number of released prostate cells can be increased by an attentive digital rectal examination (DRE) prior to urine collection. Diagnostic urine real time PCR assays have been developed recently (specific mRNAs: PCA3, AMACR, TMPRSS2:ERGa fusion gene). In our long term research program we address the question if quantitative information on selected prostate cancer biomarker mRNAs can be extracted from a heterogeneous urine cell fraction by real time qPCR. Furthermore, we explore if urine qPCR data can be used for a comprehensive non-invasive prostate tumor survey (differentiation latent/clinical tumor, malignancy, progression, metastasis, relapse, survival).

Methods: Urinary cells were isolated by filtration (50ml; n=42 urine from pathologically confirmed prostate cancer patients; n=36 urine samples from males aged <35 years as negative controls; additionally urine was collected from patients prior and after an attentive DRE). Total RNA was isolated from lysed cells (Zymo Research urine RNA isolation kit) and reverse transcribed into cDNA using anchored primers (Roche Transcriptor Kit). Quality control was performed by Lightcycler II real time SYBR Green I PCR with a 18s RNA primer pair. A geNORM and Normfinder analysis was performed with 15 housekeeping genes (PrimerDesign Ltd.) with RNA samples in duplicate reactions. A panel of 12 mRNAs known to be over-expressed in prostate cancer was quantified after correction for PCR efficiencies. GenEx software (MultiD) was used for calculations and statistical data analyses.

Results: A cell filtration assay allows highly efficient, inhibitor free total RNA isolation from spontaneous and post-DRE urine. Urine cell filtrates contain a mixture of kidney, bladder, prostate, urethral epithelial cells and leukocytes of unknown percentage composition. Hence, a relative mRNA quantification strategy has to be based on mRNAs of reference genes which are stable expressed in normal and cancer or pre- and post-DRE urine. A geNORM/Normfinder analysis identified GAPDH and CYC1 as the two best overall fitted genes for a prostate cancer urine mRNA analysis. Subsequently Lightcycler II SYBR Green assays were performed for 12 prostate cancer tumor marker genes (ABCA5, AMACR, DD3/PCA3, PCa-002, PCGEM1, PSA, PSCA, PSGR, PSMA, RPS2, TMPRSS2, TMPRSS2:ERGa). Results of relative Lightcycler real time qPCR assays with selected biomarker mRNAs will be presented.

Conclusions: Prostate cancer tumor marker mRNAs can be quantified in spontaneous and post-DRE urine of non-diseased and prostate cancer patients by real time qPCR. Multi-parameter qPCR assays might in the near future supplement the urological diagnosis (OENB P11491).

P 061

Selection of reference genes in real-time RT-PCR studies of Atlantic salmon Salmo salar.

Pål A. Olsvik1, Kai K. Lie1, Ann-Elise O. Jordal2, Tom O. Nilsen2 and Ivar Hordvik2

1National Institute of Nutrition and Seafood Research, Norway and 2Biology Department, University of Bergen, Norway

Email: pal.olsvik@nifes.no

Salmonid fishes are among the most widely studied model fish species but only limited information is available on reference gene stability in qRT-PCR studies.

The stability of six potential reference genes was examined in eight tissues of Atlantic salmon (Salmo salar), to determine the most suitable genes to be used in quantitative real-time RT-PCR analyses. The relative transcription levels of genes encoding 18S rRNA, S20 ribosomal protein, β-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), and two paralog genes encoding elongation factor 1A (EF1AA and EF1AB) were quantified in gills, liver, head kidney, spleen, thymus, brain, muscle, and posterior intestine in six untreated adult fish, in addition to a group of individuals that went through smoltification. Based on calculations performed with the geNorm VBA applet, which determines the most stable genes from a set of tested genes in a given cDNA sample, the ranking of the examined genes in adult Atlantic salmon was EF1AB>EF1AA>β-actin>18S rRNA>S20>GAPDH. When the same calculations were done on a total of 24 individuals from four stages in the smoltification process (presmolt, smolt, smoltified seawater and desmoltified freshwater), the gene ranking was EF1AB>EF1AA>S20>β-actin>18S rRNA>GAPDH. We are further evaluating the usefulness of elongation factor paralog genes as potential reference genes in Atlantic salmon. In current examinations, two new paralogs, EF1AC and EF1AD, are also studied, and the stability of these forms compared to EF1AA and EF1AB. Overall, this work suggests that the EF1AA and EF1AB genes can be useful as reference genes in qRT-PCR examination of gene expression in the Atlantic salmon.

P 062

The SPUD assay has an important role in interpretation of detecting Pneumocystis DNA in clinical specimens.

Novak T1., R.F. Miller1, A. Pooran1, M. Hoelscher2,3, M. Gerhardt2, F. Minja2, A. Zumla1, J. Huggett1

1University College London, United Kingdom, 2Mbeya Medical Research Programme, Mbeya, Tanzania and 3Ludwig Maximilian University, Munich, Germany

Email: t.novak@ucl.ac.uk

Pneumocystis pneumonia (PCP) is the most frequent AIDS-defining disease associated with an opportunistic pathogen. Identification of the causative agent, Pneumocystis jirovecii , in clinical samples requires a complicated microscopic procedure demanding considerable histological expertise. There is clear potential benefit in use of molecular methods to diagnose and monitor PCP, yet the utility of PCR remains uncertain despite the existence of substantial published work addressing this subject. One of the reasons for this uncertainty is that a negative PCR result does not always infer the absence of target ( Pneumocystis ) DNA in a clinical sample - unless that sample is assessed for the presence of PCR inhibitors. The problem lies in the fact that clinical samples may be highly heterogeneous, containing variable amounts of both host and pathogen DNA, as well as inhibitory substances which may be extracted alongside the intended target template. Inhibited specimens may be reported as ‘PCR negative’, when in fact they are ‘false negatives’ so compromise diagnostic sensitivity. It is essential to include an assessment of sample inhibition to provide confidence in interpretation of molecular detection. The SPUD inhibitor assay, using potato DNA, has been used previously to assess inhibition of PCR in cDNA samples. We have used the same strategy to measure PCR inhibition in clinical samples when assessing the presence of P. jirovecii DNA. By artificially including the SPUD amplicon in real-time qPCR reactions we have investigated whether a clinical sample is causing inhibition. We are characterising use of the SPUD inhibitor assay for routine assessment of DNA extracts from a range of clinical samples (including urine, bronchoalveolar lavage fluid and pleural fluid). This assay has the potential to provide valuable information enabling better interpretation of a negative result and aiding identification of specimens that need additional assessment.

P 063

Use of real-time RT-PCR as a substitute for nuclear run-on transcription assays.

Pennington C. J. and Edwards D.R.

University of East Anglia, United Kingdom

Email: c.j.pennington@uea.ac.uk

Tissue inhibitors of metalloproteinases (TIMPs) are a family of four multifunctional proteins with a broad range of biological activities, including inhibition of metalloproteinase activity, regulation of cell proliferation, apoptosis and the regulation of angiogenic and inflammatory responses. Elevated mRNA expression of TIMP family members correlates with malignancy and clinical outcome in many human cancer types; however, a protective role for TIMPs has also been observed in various mouse models of human cancer. Mammalian TIMP2, TIMP3 and TIMP4 genes contain 5 exons but TIMP1 has an additional short first exon which is transcribed but not translated; the translation start site is located in exon 2 and these two exons are separated by an 646bp intron 1 region that is therefore unique to TIMP1 genes. Previous studies have shown that the control of TIMP1 expression occurs at the level of transcription with regulatory elements present in the promoter region and in the first intron prior to the translational start site (Dean et al, J. Biol. Chem. 2000:275 p32664-32671). We demonstrate using nuclear run on assays and a novel TaqMan based analysis to quantify transcription levels, that Timp1 transcription following stimulation of 10T1/2 mouse fibroblast cells by PMA and growth factors is not uniform across the gene. Real Time PCR probes, located in different introns throughout Timp1, revealed a marked decrease in gene transcription of intron 1 after +570. Analysis using 3’-RACE further indicates the presence of primary transcripts that terminate at various sites towards the 3’-end of intron-1. These data indicate that Timp1 but not other Timp family members, is subject to control by transcriptional attenuation within its distinctive intron1 region. Further work will concentrate on the mechanism of transcription attenuation and the possible role played by chromatin remodeling.

P 064

High Resolution Melt analysis with non-saturating dyes.

Jason Barrett, Paul Kayser, Natalie Simpson, Winnie Chong, Mark Stevens

1Quantace, United Kingdom

Email: info@quantace.com

High resolution melt (HRM) analysis is a rapidly developing technique for mutation detection based on the temperature dissociation characteristics of DNA. Development of the technique has relied on the continued evolution of fluorescent binding dyes. These "Third-Generation" dyes include LC Green+, SYTO9 and EvaGreen. Previous comparative experiments of these dyes with SYBR Green I identified the importance of saturating dye concentrations in reducing dye redistribution. It has been postulated that dye redistribution leads to a decrease in sensitivity and therefore reduced confidence in mutation calling. We have shown that LC Green+, SYTO9, EvaGreen and SYBR Green I are not saturating at concentrations non-inhibitory for PCR. EvaGreen, although non-saturating, successfully detects class IV mutations. These results lead to the conclusion that dye-chemistries, and not saturation per se are important principles that need to be addressed for successful HRM analysis.

P 065

Gene Expression Signature in Peripheral Blood Detects Thoracic Aortic Aneurysm.

Samaha R.

Applied Biosystems, United States of America

Email: samaharr@appliedbiosystems.com

Thoracic aortic aneurysm (TAA) is usually asymptomatic and associated with high mortality. Although adverse clinical outcome is preventable by surgical repair, identifying at-risk individuals is difficult. Our goal was to identify a potential gene expression signature in peripheral blood that may allow development of noninvasive screening tests to identify individuals at risk for TAA disease.

Gene expression profiles of peripheral blood samples collected from 58 individuals diagnosed with TAA and 36 normal individuals (controls) were analyzed using the Applied Biosystems Expression Array Systems and Human Genome Survey Microarrays. SAM (Significance Analysis of Microarray) analysis of 29,098 RNAs identified 1199 candidate signature genes (markedly up- or down-regulated) characterizing the TAA disease. Gene classification and biological pathway analyses of these signature genes revealed potential molecular mechanisms underlying this disease. Signature genes were also determined to delineate ascending TAA vs. descending TAA, as well as TAA with or without family history.

Additionally, 41-gene prediction models for risk assessment of TAA were built for all-gender and male or female, respectively, using a training set containing 36 TAA patients and 25 controls. 10-fold cross-validation on these prediction models yield 82%, 90%, and 97% overall accuracy for each model, respectively. When these prediction models were applied on an independent testing sample set containing 22 TAA patients and 11 controls, the overall prediction accuracies for all-gender, male and female models were 79%, 70% and 77%, respectively.

This study provides a comprehensive gene expression profile of peripheral blood cells from thoracic aortic aneurysm patients and normal individuals. The biological pathways associated with the signature genes of TAA and its subtypes may provides further insights into the molecular pathogenetic mechanisms attributed to the pathogenesis of this disease. Our results also demonstrated a distinct RNA “signature” of aneurysm disease, setting the stage for a blood-based gene expression test may to facilitate risk assessment and early detection of thoracic aortic aneurysm disease.


Session:   High throughput quantitative PCR

Poster number   P 066    P 076

Location:           Student Cafeteria

P 066

Expression profiling of receptor-like cytoplasmic protein kinases (class VI) of Arabidopsis.

Manuela Elena Jurca, Attila Feher

Institute of Plant Biology - Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary

Email: elenaj2006@yahoo.com

Understanding the mechanisms by which plant perceive environmental signals in order to activate adaptive responses is of fundamental importance to biology. Receptor-like cytoplasmic protein kinases (RLCKs) are plant specific proteins encoded by more then 150 different genes in the Arabidopsis genome. Despite of their high number, the available information on the potential function of RLCKs is very limited. In this report, the analysis of the gene expression pattern of 14 members of one of the RLCK families (RLCK class VI) is described. The technique of real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to determine the transcript levels in Arabidopsis seedlings under a series of abiotic stress/hormone treatments as well as in different organs. Quantitative gene expression data was normalized for the expression of GAPDH-2 and actin (Act2/Act8) genes. In order to help the interpretation of the results, a stress responsive marker gene, RD29A, was also determined. Based on the expression data it can be concluded that the biological functions of RLCK proteins, despite their structural similarity, might be highly divergent and is regulated, at least partly, at the transcriptional level.

P 067

LightCycler® 480 Real-Time PCR System: Innovative Solutions for High Throughput PCR.

O. Geulen, G. Tellmann

Roche Diagnostics, Roche Applied Science, Germany

Email: Oliver.Geulen@Roche.com

The LightCycler® Real-Time PCR Systems from Roche Applied Science have set standards for maximum flexibility, high speed and outstanding data accuracy. The latest innovation, the LightCycler® 480 Real-Time PCR System, continues this tradition and extends it to higher throughputs (96-well / 384-well format) by using plate-based analysis format. The modern instrument design, outstanding technical and software features, as well as advanced reagents and disposables of the LightCycler® System pave the way for high-performance real-time PCR analysis. The LightCycler® 480 software provides versatile solutions for the most common real-time PCR applications like gene detection, gene expression and genotyping analysis. The system’s basic software package comprises basic software and application-specific modules which provide diverse methods for innovative analysis workflows. Furthermore, the system now offers a new analysis tool for high-resolution melting curve analysis (HRM), providing an innovative method to scan genes for unknown variations.

P 068

Mutation Scanning Using the LightCycler® 480 System.

C. Weilke, J. Hurlebaus, L. Gretschel, C. Roessler

Roche Diagnostics, Roche Applied Science, Germany

Email: Christian.Weilke@Roche.com

Established methods for PCR genotyping using dye-labeled probes only allow for the detection of an expected sequence variation forming a mismatch to the probe sequence. For screening for any mutation in longer sequence intervals, sequencing is state of the art. However, sequencing is still expensive and not suited for high-throughput analysis.

Here, we describe a method that enables scanning of genes for sequence variations by

high-resolution melting analysis of amplicons. The equipment required comprises of a real-time PCR instrument enabling high resolution melting curve analysis like the LightCycler® 480 Instrument, a pair of target-specific PCR primers, PCR reagents containing a special fluorescent dye for detection, and an adequate evaluation software.

After PCR, the amplicons from several samples (gene sections of up to 600 bp) are analyzed by high-resolution melting. For amplicons with sequence variations, the melting curves differ in shape, especially when heterozygote DNA has formed heteroduplices. The new software module analyzes these small differences and groups samples of similar curve shape. Using samples of known sequence as standards, other samples in the same group can be assigned to this sequence.

The new method enables detection of any sequence variation in the amplified DNA section. It is relatively cheap, requires little optimization (only a specific primer pair for amplification using the universal PCR master), and facilitates high-throughput screening of samples in 96- or 384-well plate formats.

Therefore, the LightCycler® 480 System is extended by a PCR reagent kit containing a new fluorescent dye that is optimized for this application, and a software module for high-resolution melting curve analysis.

P 069

New concepts for accelerated real-time PCR analysis.

Thorsten Traeger1, Kirsten Hildebrand1, Dirk Loeffert1, Ralf Peist1, Annette Tietze1, Andrea Stutte-Rabe1, Ulla Deutsch1, Dile Holton2, and Andreas Missel1

1QIAGEN GmbH, Hilden, Germany and 2QIAGEN Sciences, Germantown, US

Email: thorsten.traeger@qiagen.com

Protocols for fast PCR provide an effective means of increasing assay throughput and significantly reducing the time required to go from nucleic acid template to final result. However, fast PCR using standard PCR chemistries has until now suffered from reduced sensitivity as well as increased variability. We will focus on how to overcome the challenges of achieving fast PCR, both in real-time PCR and RT-PCR analyses and in end-point PCR applications. Issues that will be discussed include:

• The use of optimal combinations of cations and additives in the PCR buffer to provide specific and accelerated annealing of primers and probes to nucleic acid templates.

• The use of a recently developed hot-start DNA polymerase and inhibition-relieving agents to improve fast end-point PCR assays and fast real-time RT-PCR assays (both one-step and two-step) which previously exhibited poor sensitivity and specificity.

Data showing successful real-time and end-point PCR analyses with time savings of up to 70% will be presented. The real-time PCR data will include data generated using different detection formats, such as SYBR Green dye and sequence specific probes.

P 070

On-Chip Polymerase Chain Reaction with integrated Real-Time Detection.

J. Felbel1, A. Reichert1, S. Julich1, M. Kielpinski1, M. Urban1, N. Häfner2, M. Dürst2, J. Weber3, and T. Henkel1

1Institute for Physical High Technology, Albert-Einstein-Str. 9., D-07745 Jena, Germany, 2Clinic for Obstetrics and Gynecology, Friedrich-Schiller-University Jena, Bachstr. 18, D-07740 Jena, Germany and 3Analytik Jena AG, Konrad-Zuse-Str. 1, D-07745 Jena, Germany

Email: anett.reichert@ipht-jena.de

The mechanistic simplicity of the PCR process made it an ideal candidate for automation and miniaturization. The advantages of miniaturized PCR-chip devices over conventional thermocyclers are low power consumption, fast total analysis time, and reduced amount of sample and reagents. During the development of micro chip thermocyclers for on-chip PCR several strategies have been followed. Of particular importance thereby have been the stationary and continuous flow PCR chips [1]. The fabrication of PCR micro chips is based on the micro system technology for its ability to produce small structures. Importantly, the micro fabrication technique also provides a possibility for integrating various functional components on the same chip. The aim of PCR on-chip developments at our institute (IPHT) is the integration of fluidic and thermal management with optical readout for real-time detection [2]. An important point of real-time detection is the specific detection and the possibility for quantification of PCR products on-chip. We will show the detection system and the results of real-time detection on the developed stationary working 2D PCR chip. We used Sybr-Green and double-labeled fluorescent TaqMan Probes (FAM/TAMRA) to detected the fluorescent signals. So it was possible to reach a limit of detection of on-chip PCR by 10 DNA molecules in a sample volume of 1 µl.

The vision of another special research project is the establishment of a system to detect single disseminated tumour cells in the blood of patients with cervical carcinoma. A special characteristic of these cells is the expression of oncogenes encoded by Human Papilloma Viruses (HPV), which can be specifically amplified by in-situ RT-PCR [3]. Therefore, we have developed a micro chip reactor for application of flow-through in-situ RT-PCR in tumor diagnostics. One step of device development is the transfer of the real-time detection system from the stationary chip to the flow-through PCR chip. The general suitability of the chip system was proven by RT-PCR of RNA, isolated from cells, expressing the HPV 16 target oncogene and will be present

P 071

Optimisation of a Real Time RT-PCR protocol for the analysis of gene expression in hop tissues.

Lina Maloukh 1,2, Jelle De Keukeleire 1,2, Erik Van Bockstaele 1,2 , Isabel Roldán-Ruiz 1

1Institute for Agricultural and Fisheries Research, Unit Plant, Applied Genetics and Breeding, Caritasstraat 21, 9090 Melle, Belgium and 2Ghent University, Faculty of Bioscience Engineering, Department of Plant Production, Coupure Links 653, 9000 Ghent, Belgium

Email: Lina.Maloukh@ilvo.vlaanderen.be

Hop (Humulus lupulus L.) is a dioecious, perennial, climbing plant, included in the family Cannabaceae. Female hop plants are cultivated in most temperate regions of the World, for the extraction of secondary metabolites. Important secondary metabolites produced by hop include α-acids and β-acids and essential oils, which are used in beer brewing, as well as the prenylated chalcones, Xanthohumol, and Desmethylxanthohumol, which exhibit interesting bioactive properties. All these compounds are synthesized in relatively high amounts during the development of the female inflorescences of hop into cones and are accumulated in the lupulin glands. The most prominent hop prenylflavonoids are the prenylchalcones Xanthohumol and Desmethylxanthohumol.These prenylchalcones can be readily converted to isomeric prenylflavanones, whereby Xanthohumol gives rise to Isoxanthohumol and Desmethylxanthohumol renders a mixture of 8-prenylnaringenin and 6-prenylnaringenin. Xanthohumol displays a broad spectrum of inhibition mechanisms at the initiation, promotion and progression stages of carcinogenesis. 8-prenylnaringenin, which results from isomerization of Desmethylxanthohumol in the brew kettle, is the most potent phytoestrogen known to date. It is, therefore, of great interest to identify and characterize the genes that regulate the formation of Xanthohumol and Desmethylxanthohumol in female hop plants. In a previous study (De Keukeleire et al. submitted) we used the differential screening technique cDNA-AFLP to identify candidate genes involved in the biosynthetic pathway of prenylflavonoids in hop. However, up to now, only semi-quantitative expression profiles are available for these candidate genes. Therefore, a detailed expression analysis of the candidate genes in different hop tissues and at different developmental stages using Real-Time RT-PCR is essential to demonstrate the possible implication in these genes in the biosynthetic pathway of prenylchalcones in hop. Our Objective in this study was to develop a test for the analysis of the expression profiles of previously identified candidate genes in hop tissues. Here we report on the selection of suitable housekeeping genes and on the expression levels of a putative transcription factor HEN1 homologue in different hop tissues from several genotypes, using Real Time RT-PCR.

P 072

Q-PCR analysis of the effect of hepatocyte growth factor on Doxorubicin induced cell death.

Salehi M., Keyhanian K., Salehi R.

Dept. of Genetics and Molecular Biology, Medical School, Isfahan University of Mdical Sciences, Isfahan, Iran

Email: m_salehi@med.mui.ac.ir

Hepatocyte Growth Factor (HGF/SF) has opposite biological activities in regulating apoptosis, also molecular mechanisms involved in these processes are not clearly defined. We investigated HGF ability to protect cell death which was induced by a DNA damaging agent (Doxorubicin). Also Survivin and XIAP mRNA levels were compared in HGF treated and not-treated cells.

Cell proliferation and death were assessed using MTT assay and dye exclusion tests. Quantitative real-time PCR was used to evaluate Survivin and XIAP expression levels after treatment with HGF. ELISA was performed to quantify HGF secretion in selected cancer cell lines media.

HGF appeared to have inhibitory effect on Doxorubicin induced cell death in all cell lines we tested. It had minimal effect on XAIP and Survivin expression levels in MRC-5, MOLT-4 and AGS cell lines; except for XIAP expression level in AGS cell line which was increased substantially after treatment. Surprisingly, in KG-1 cell line, XIAP and Survivin expression levels were significantly reduced after HGF treatment.

Although several members of IAP gene family are reported to play role in HGF induced antiapoptotic pathway, we showed that XIAP and Survivin do not seem to be involved.

P 073

qPCR Arrays-based gene profiling of Tie2-expressing monocytes (TEMs): contamination analysis by qPCR and multiple linear regression analysis of data.

Pucci F. 1,2,4 Venneri MA. 1,2 Moi D. 1,2 Di Serio C. 3,4 Naldini L. 1,2,4 De Palma M. 1,2

(1) San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Via Olgettina 58, 20132, Milan, Italy; (2) Angiogenesis and tumour targeting research unit (ATTRU), Via Olgettina 58, 20132, Milan, Italy; (3) University Centre for Statistics in the Biomedical Sciences (CUSSB), Università Vita-Salute San Raffaele, Via Olgettina 58, 20132 Milan, Italy; (4) Università Vita-Salute San Raffaele, Via Olgettina 58, 20132 Milan, Italy

Email: ferdinando.pucci@hsr.it

Tumours are made not only of neoplastic cells, but also of different types of stromal cells, which play both anti- and pro-tumour activities during cancer progression. The concept that haematopoietic cells – myeloid-lineage cells in particular – are more likely to promote tumour growth than to mount effective anti-tumour responses has received broad recognition. However, little is known of the molecular mechanisms responsible for the pro-tumour activities of myeloid cells. We recently described a population of bone marrow-derived monocytes that express Tie2, the angiopoietin receptor previously known to be specifically expressed by endothelial cells (ECs). TEMs specifically home to tumours and other angiogenic sites and are required for tumour angiogenesis and growth, but are distinct from endothelial progenitor cells since they do not incorporate into growing vessels. The precise identity and the molecular bases of the pro-angiogenic activity of TEMs still need to be elucidated.

In this study we compared the gene expression profile of TEMs with that of other myeloid cells, isolated from both tumours and non-neoplastic tissues of Tie2-GFP reporter transgenic mice, by TaqMan Low Density Arrays. These devices are 384-wells custom cards configured as 93+3 target/housekeeping genes in quadruplicate. Since both TEMs and ECs share phenotypical features and express GFP in our model, the potential ECs contamination of flow-sorted TEMs represents an important technical issue. We used qPCR to quantify ECs contamination by assessing the expression of 5 ECs-specific genes. We then designed 3 arrays containing about 300 genes among cytokines and growth factors, receptors, proteases, guidance and adhesion molecules, immunity-related genes and transcription factors. Data obtained from 2 biological replicates (each made by cells harvested from 10-15 mice) were analysed with a multiple linear regression model to identify differentially expressed genes.

We were able to virtually abolish ECs contamination by inlcuding a negative selection to exclude ECs during TEMs isolation. Our results indicate that TEMs are a defined population of tumour-infiltrating myeloid cells, since 33% of the interrogated genes were differentially expressed in TEMs versus GFP-neg tumour macrophages (p<0.05). Greater differences in gene expression were found between TEMs and peritoneal macrophages and between TEMs and CD11b+Gr1+ myeloid-derived suppressor cells. Some of the differentially expressed genes may have a critical role in angiogenesis and tissue remodelling; moreover, TEMs express a unique set of immuno-modulator molecules, suggesting that TEMs may also create an immune-privileged environment that promotes tumour growth and ECs survival.

These studies will be extended to a genome-wide profile on human TEMs. These results will help elucidating the biology of TEMs in mice by focusing on the relevant targets and will potentially identify novel targets of anticancer drugs.

P 074

Transcriptional profiling of the ABC transporters family using Taqman Low Density Array.


ICSN - CNRS, Gif-sur-Yvette, France

Email: Eric.Jacquet@icsn.cnrs-gif.fr

The ATP-binding cassette (ABC) transporters, a large family of transmembrane proteins, have essential physiological and protective functions in cells. ABC transporters are encoded by 49 identified genes in human and have been classified in seven subfamilies (A to G). Mutations in genes encoding these proteins are related to diverse genetic diseases in human. Most of these ABC proteins are involved in transport of a large variety of substrates (amino acids, lipids, ions, sugars, drugs...) across cellular membranes. Some ABC transporters have essential functions such as excreting liver or kidney toxins, or limiting penetration of toxic molecules in vital organs, such as brain or placenta. Multidrug resistance of cells is also correlated with the overexpression of several ABC transporters that induce drug-resistance phenotypes of tumor cells to anti-cancer chemotherapies.

To carry out ABC expression studies, we have developed high throughput quantitative PCR using TaqMan probes and ABI 7900HT QPCR instrument with fully automated robotic loading. We have designed microfluidic cards (TLDA, TaqMan Low Density Array, applied Biosystems) that allow quantifying simultaneously the expression of all these ABC genes in human or in mouse. These TLDA also contain 10-15 housekeeping genes for data normalizations. QPCR efficiency of the probes was first controlled using samples standard curves, plasmid dilutions and linear regression calculations (LinRegPCR). Housekeeping genes were selected with GeNorm and NormFinder software. Normalization was provided using the geometric mean of 3-5 selected genes. Results were expressed with the relative quantification using 2-ddCt method. Moreover, in the cell lines studies, the copy numbers of mRNA per cells were estimated to have a better overview of the ABC expression pattern.

This technique allow us to get an overall picture of the expression level of the ABC transporters, in various organs, tissues and cell lines from human and mouse. We analyzed the ABC expression pattern of different cell lines commonly used for cytotoxicity assays. We compared these expression levels to their resistant sublines and we correlated ABC profiles with the chemoresistance phenotypes. We have also mapped the expression of ABC transporters in different mouse organs and used this in-vivo model to measure their variations in response to a cytotoxic injection. Finally, we have studied the ABC profiles 1) in human liver at different stages of development (foetal, new-born, adult) and 2) in biopsies of patients with acute leukemia in correlation with their sensitivity towards chemotherapies.

To date, several ABC transporters do not have any known substrate and physiological function yet. Therefore, studying expression of this gene family may help to explain their potential role in human diseases and anti-cancer therapies.

P 075

BioMark™ Dynamic Arrays and Digital Arrays for Gene Expression Quantification and Absolute Quantification of mRNA in Human Saliva Samples.

Pieprzyk Martin, Yaccato Karin, Herren Michael

Fluidigm Corp., United States of America

Email: marc.unger@fluidigm.com

Human saliva samples hold great promise for a simple and direct method to obtain gene expression profiles and detect cancer biomarkers. Messenger RNA sequences from human saliva samples were quantified extremely accurately using digital PCR. The BioMark Dynamic Array performs qPCR on 48 genes against up to 48 samples. The dynamic array was used to profile gene expression on a panel of 48 genes for each sample.

P 076

Use of standardized mixtures of internal standards in automated high throughput quantitative PCR to generate quality-controlled transcript abundance measurements that are reproducible across institutions.

Elizabeth Herness Peters, James C. Willey, John C. Anders, Charles R. Knight, Bradley J. Austermiller

Gene Express Inc., 975 Research Drive Toledo, Ohio, USA, University of Toledo Health Sciences Campus Toledo, Ohio, USA

Email: ehpeters@geneexpressinc.com

Profiling mRNA transcript abundance (TA) of multiple genes in whole blood or biopsy samples vastly increases the potential for phenotypic characterization over that obtainable by morphological analyses. It is widely anticipated that a TA measurement method that generates data suitable for regulatory review will facilitate development of new drugs and improved diagnostic tests. Standardized RT-PCR (StaRT-PCRTM) has the performance characteristics recommended by the FDA to generate such data. These performance characteristics are achieved by measuring each gene relative to a known quantity of its respective internal standard formulated within a Standardized Mixture of Internals Standards (SMISTM). In order to determine the inter-site precision of TA measurements, fifteen genes were analyzed over more than a two year period in multiple reverse transcribed cDNA samples from the same lot of Stratagene Universal Human Reference RNATM at the Standardized Expression Measurement (SEMTM) Center established at the University of Toledo (UT: Toledo, OH) and two newly established SEM Centers located at Gene Express (GX: Toledo, OH) and Gene Logic (GL: Gaithersburg, MD). Potential sources of analytical variation included variation across different commercial liquid handlers (Packard and Hamilton), two thermocyclers (MWG and Biorad), two Caliper devices (AMS 90 SE30 and LC90 models) with multiple chips, and cDNA samples from two different aliquots of the same lot of RNA sample across three different sites. The measured genes spanned an expression range from 37 GPT transcript molecules/10^6 ACTB transcript molecules to 99,000 MYC transcript molecules/10^6 ACTB transcript molecules. When the average data from GX, GL and UT were compared, for each gene the range of TA measurements was less than two-fold with CVs ranging from 10.9% to 29.6%. SLC15A2 and GPT had larger CVs in part due to stochastic sampling variation associated with low numbers of molecules (fewer than 100 molecules) in each assay. Variability was higher across sites (e.g. UT vs. GL) than within sites. This study demonstrates that use of SMIS reagents and StaRT-PCR technology generates TA data that may be directly compared at different sites over time. This will enable generation of a standardized, numerical database, which is essential for wide-scale implementation of PCR in molecular diagnostic tests and drug development.


Session:   qPCR NOS Session

Normalization & Optimization & Standardization

Poster number   P 077    P 104

Location:           Student Cafeteria

P 077

EasyBeacon™ -New probes ideal for Real Time PCR detection of methylation status of single CpG duplets and SNPs.

Christensen U.B., Nielsen C. & Skadhauge K.

PentaBase Aps, Denmark

Email: ubc@pentabase.com

The invention of real time PCR has revolutionized the way of determining the expression status of a gene, in a fast and precise manner. Detection and quantification of the target can be done by using different technologies, among others molecular probes. One of the major challenges for the molecular probes is to be able to discriminate between very similar target varying in as little as one nucleotide, e.g detection methylation status of single CpG duplets and SNP detection.

The EasyBeacons™ presented here are based on the novel technology of Intercalating Nucleic Acid, INA®, linked to a fluorophore and a quencher. INA® is composed of normal DNA nucleotides and Intercalating Pseudo Nucleotides (IPNs). The fact that the EasyBeacons™ are mostly composed of normal DNA nucleotides means, that in many respects the EasyBeacons™ behave like DNA based probes, allowing the use of standard buffers, primers and enzymes and hence reduces the optimisation efforts.

The IPNs of the EasyBeacons™ comprise hydrophobic moeities that will bring the fluorophore and the quencher of unbound probe in close proximity of each other. The quenching of the flourescense, and the improved signal-to-noise ratio are therefor not dependent on the sequence of the DNA nucleotides. Due to the high affinity of INA® towards complementary DNA, the EasyBeacons™ can be made shorter and hence more specific than their DNA counterparts.

The non temperature dependent quenching EasyBeacons™ is also very suitable for multiplex PCR , and compatible with most, if not all the commercially available real time PCR instruments.

The results presented here show the use of EasyBeacons™, generating a better signal-to-noise ratio, a higher affinity and specificity in detection of the methylation status of single CpG duplets and SNPs.

Furthermore, taking advantage of the possibility to include an end point verification measurement, since the EasyBeacons™ are not degraded during PCR amplification, is proven to be valuable.

The ease of design and optimization of the real time PCR assays using the EasyBeacons™ is also discussed.

P 078

Effect of different dietary fiber on the expression rate of the inflammatory marker genes TGF β and TNF α, respectively NFkB in the gastrointestinal tract of weaning piglets.

Karl Schedle1, Michael W. Pfaffl2 and Wilhelm Windisch1

1Division of Animal Food and Nutrition, Department of Food Science and Technology, University of Natural Resources and Applied Life Sciences, Austria and 2Institute of Physiology, TUM Weihenstephan, Germany

Email: karl.schedle@boku.ac.at

In the present study we investigated the effect of two fiber sources differing in microbial degradability on expression rate of the anti- and pro-inflammatory marker genes TGF β and TNF α, respectively, the transcription factor NFkB in the gastrointestinal tract (GIT) of weaning piglets. The study employed a total of 36 weanling piglets fed ad libitum. Two diets were modified by adding wheat bran or pollen from Chinese Masson pine (pinus massoniana) and compared to a control group. The amounts of added fiber sources were adjusted to result in equal contents of total dietary fiber. Animals were distributed according to litter, sex and initial body weight among the 3 types of diet. Tissue samples of the (GIT) were collected and stored at -80 °C. Total RNA of samples was isolated using TriFast. qPCR was carried out as two-step PCR. Relative quantification of cDNA (respectively, mRNA) was carried out with the Eppendorf realplex Cycler. The crossing points were acquired with the delta-Ct-method using the mean expression of two reference genes (histone H3 and beta-actin). Relative quantification was calculated by the delta-delta-Ct method, compared to the untreated control group. Gene expression rate of the pro-inflammatory marker Gene TNF α did not differ between fiber sources in the gut. In mesenterial lymph nodes, however, TNF α was up-regulated by wheat bran (n.s) and by pine pollen (p<0.1). Regarding the anti-inflammatory marker gene TGF β, wheat bran resulted an up-regulation in the stomach (p<0.05) and jejunum (p<0.05), while in the ileum and colon gene expression did not differ from control (n.s.). Pine pollen addition resulted in numerical up-regulation in stomach and jejunum (n.s.) while in ileum and colon the reactions were of minor extent. In mesenterial lymph nodes, however, expression of TGF β was numerically up-regulated by pine pollen (n.s.) while no effect was visible, with wheat bran. Only in the stomach, NFkB was numerically up-regulated by wheat bran and pollen (n.s.). The results indicate the ability of fiber sources to affect the expression of inflammatory marker genes TGF β and TNF α. The simultaneous up-regulation of both pro- and anti-inflammatory marker genes in mesenterial lymph nodes e.g. in case of the pine pollen group seem to reflect a general stimulation of activity of the immune system through additional dietary fiber intake. This hypothesis is supported by the absence of reaction of the pro-inflammatory marker gene TNF α in the small and large intestine. This hypothesis is furthermore confirmed by the absence of up-regulation from NFkB, a transcription factor for TNF α and the simultaneous up-regulation of anti-inflammatory marker gene TGF β in other gastrointestinal tissues. The up-regulation of TGF β in the stomach and jejunum may therefore be interpreted as indicator of increased cell proliferation.

P 079

Effects of different SybrGreen master-mix manufacturing lots and suppliers on real-time PCR parameters.

Barbara Rocca, Silvia Calatroni, Ilaria Giardini, Marina Boni, Irene Dambruoso, Paolo Bernasconi

Division of Hematology, IRCCS Policlinico S. Matteo Foundation, Pavia, Italy

Email: s.calatroni@smatteo.pv.it

Real-time polymerase chain reaction (PCR) methodology is now broadly used to quantitate target sequences for diagnostic purposes. Despite the growing availability of commercial in vitro diagnostic assay, “home-made” real-time PCR methods, using commercial reagent master-mix are still applied in many laboratories. Aim of the present study was to analyse the effect of different SybrGreen master-mix manufacturing lots and suppliers on real-time PCR parameters. The analysis was conducted on a well-assessed “home-made” relative quantification BCR-ABL assay, using ABL as normalising gene and five log dilution of leukemic cell line K562 cDNA. To minimize variability due to reverse-transcription (RT), all the cDNA was obtained through the same RT reaction using random hexamers primers and SuperScript II (Invitrogen). We compared four different manufacturing lots of a same product (same p/o number), provided by a single supplier (Applied Biosystems) and six different suppliers of SybrGreen master-mix, with Rox (Applied Biosystems, Bio-Rad, Eurogentec, Finzyme, Qiagen and Stratagene). All the reaction were conducted in duplicates on a GeneAmp5700 (Applied Biosystems), using the same production lot of primers and the same thermal conditions. As expected, differences in PCR parameters has been, in some cases, observed in master-mixes of different suppliers, due to differences in reagents composition. Interestingly, differences in Tm values (max deltaTm=1.5°C), Ct values (for each dilution point deltaCt range between 0.01 and 1.45 for BCR-ABL , and between 0.06 and 2.07 for ABL), max deltaRn at the last PCR cycle and reaction efficiency have been also evidenced when comparing different lots of the same product; efficiency variations were not always comparable in the BCR-ABL and ABL assays. In conclusion, if master-mix composition is surely important during assay optimisation, particular care has to be taken introducing different lots of a same commercial master-mix, especially in diagnostic assay, and validation of the new preparation lot should be advisable.

P 080



12nd Dept. of Pathology and 21st Dept. of Surgery, Semmelweis University, Budapest, Hungary

Email: cac@korb2.sote.hu

INTRODUCTION  Members of the claudin gene family are differently expressed in healthy tissues and various types of tumours. According to the literature the investigation of claudin patterns lead to unequivocal results. Therefore, we examined the expression of 9 claudin types in invasive ductal breast carcinomas and their normal adjacent tissue (NAT).

METHODS  A total of 23 breast samples (8 invasive ductal carcinomas, their 8 NATs, 3 lobular carcinomas, and 4 NATs) have been analysed for their claudin pattern with real-time PCR and immunohistochemical method.

Sybr Green real-time PCR method was used for detection of claudin-1, -2, -3, -4, -5, -7, -8, -10 and -12. A melting peak profile was produced after each amplification. The PCR data were analysed with both the Relative Expression Software Tool XL Version 2 (REST-XL) and the latest version of REST-384 using pair wise fixed reallocation randomisation test.

Immunohistochemical detection (streptavidin-peroxidase procedure) of claudin-1, -2, -3, -4, -5 and -7 was performed on formalin-fixed, paraffin-embedded sections on a set of 20 invasive ductal breast carcinomas.

RESULTS  The mRNA expression of claudin-1, -2, -3, -4, -5, -8 and -10 were found to be down-regulated in all tumours, while claudin-7 and -12 were up-regulated in carcinomas compared to their NATs. Statistically significant alteration of claudin-12 was observed in the ductal type of invasive carcinoma group only (with a p value of 0,001).

Immunohistochemistry revealed the expression of claudin-1 as being markedly decreased in tumours compared to NATs. Immunohistochemically, there were no significant differences in claudin-2, -3, -4 and -7 expression in the majority of invasive ductal breast carcinomas as compared to NATs, in concordance with the real-time PCR data. Claudin-5 was not only observed in tumour cells, but in endothelial cells as well. Claudin -8, -10 and -12 immunohistochemistry is being adjusted.

CONCLUSION  According to our observations, the expression of claudin-1, -7 and -12 seem to be different in tumours compared to normal breast tissue, as demonstrated by real-time PCR and immunohistochemistry. This finding may suggest their possible role in carcinogenesis. The significantly higher expression level of claudin-12 requires further investigation.

P 081

Expression of pro-inflammatory cytokines, chemoattractant proteins and their receptors in bovine follicles before and after GnRH application and ovulation.

Kliem H., Schams D., Berisha B.

Physiology, TUM, Germany

Email: heike.kliem@wzw.tum.de

The rupture of a follicle during ovulation has been thought to be an inflammatory like process with the induction of pro-inflammatory cytokines and invasion of immune cells. The aim of this study was to evaluate the expression pattern of tumor necrosis factor alpha (TNFalpha), its two receptors (TNFalpha-R1, TNFalpha-R2), interferone gamma (INFgamma), interleukine-1 beta (IL-1beta), monocyte chemoattractant protein-1 (MCP-1), its receptor CCR-2, eotaxin-3 and its receptor CCR-3 in time-defined follicle classes before and after GnRH application and after ovulation in the cow. Ovaries containing preovulatory follicles or new corpora lutea (CL) were collected at approximately 0, 4, 10, 20 and 25h (follicles) and 60h (new CL) relative to injection of GnRH to induce an LH surge (n = 5 animals per group). The mRNA expression was evaluated by a two step real time PCR (Rotor-Gene 3000) and the data were normalised with the mean value of the three housekeeping genes ubiquitin, GAPDH and histone. TNFalpha, its two receptors and INFgamma showed no regulation, whereas IL-1beta revealed an up-regulation at 25h (around ovulation) and 60h (new CL). The expression of MCP-1 was increased during the LH surge (4h) and further on around ovulation (25h) and in the new CL (60h). Its receptor CCR-2 was down-regulated 10h after GnRH application and increased again till its maximum expression at 60h. In contrast to MCP-1 showed eotaxin-3 its highest expression at 0h and 4h (LH surge) followed by a decrease at 20h and 25h (around ovulation) after GnRH injection. Its receptor CCR-3 was decreased around ovulation (25h) and increased in the new CL (60h). These data suggest that during ovulation only IL-1beta and MCP-1 seem to play an important role. The increased expression of eotaxin-3 during the LH surge could trigger the invasion of eosinophils into the ovulating follicle, which might be necessary for an optimal angiogenesis in the developing CL.

P 082

External cell control quantitative RT-PCR (eccPCR): a new technique for reliable detection of subtle changes in mRNA expression.

Polett Ribiczey 1, András Bors1, Gabriella Köblös2, Anna Brózik 1, Zsuzsanna Ujfaludi 3, Mária Magócsi 1, András Váradi 2, Attila Tordai 1, Tünde Kovács 1, Tamás Arányi 2

1National Medical Centre, Institute of Haematology and Immunology, Budapest, Hungary, 2Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary and 3University of Szeged, Faculty of Sciences, Department of Biochemistry and Molecular Biology, Szeged, Hungary

Email: ribiczey@kkk.org.hu

Quantitative RT-PCR (qRT-PCR) is a widely used method to determine relative gene expression levels. Quantification of the observed expression levels becomes reliable after normalization to the expression of an internal standard gene. However, the expression of commonly used internal standard genes is often unstable, which considerably bias quantification. To overcome the drawback of unstable internal standards, we developed a new method, called external cell control PCR (eccPCR). This method is based on the addition of control cells to the studied cells before RNA extraction and qRT-PCR. Only the control cells express the reference gene, while only the studied cells express the gene of interest. Here we present the validation of the method in various model systems including both adherent and non-adherent studied cells and either mammalian or Drosophila control cells. We demonstrate that in contrast to the use of common internal standard genes the eccPCR technique allows accurate quantification of small expression level differences of the genes of interest.

P 083

External standard curve for absolute quantification of genomic DNA sequences by Real Time PCR.

Raffaele Di Francia1, Ferdinando Frigeri1, Rosaria De Filippi1,2, Giancarla Iaccarino1, Gennaro Varriale3, Antonio Arbitrio3 and Antonio Pinto1

1Hematology-Oncology Unit, Istituto Nazionale Tumori, Fondazione "G. Pascale", IRCCS, Naples, Italy, 2Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli Studi 'Federico II', Naples, Italy and 3Senebgene s.r.l., Naples, Italy

Email: rdifrancia@libero.it

Background. Real-Time PCR is the method of choice for quantification of a given DNA target. Determination of the relative abundance of a DNA sequence is usually accepted as a reliable tool to measure differences among samples. However, absolute determination of the number of copies for a DNA target can result of a greater value for minimal residual disease (MRD) evaluation in hemopoietic neoplasms.

Methods. We describe a simple method to build an external standard curve by means of a recombinant plasmid DNA, exactly quantified by competitive PCR against a specific competitor, i.e. a non-human DNA fragment of known concentration. Such approach was developed to quantify two genetic marker breakpoints of non-Hodgkin’s Lymphomas, i.e. the t(14;18) and t(11;14), respectively associated to Follicular (FCL) and Mantle Cell (MCL) lymphomas. Quantitative-PCR (Q-PCR) assay was performed by two different methods: i) TaqMan chemistry for t(14;18) and ii) SYBR Green for t(11;14). Standard stocks were constructed by diluting 900000.0 copies of translocation-specific plasmids with 500 ng of DNA from a pool of healthy donors. Standard stocks were further serially diluted (900000.0-9.0) to build external standard curves for each of the targets, i.e. FCL curve (pFCL) and MCL curve (pMCL). Copy numbers of t(14;18) or t(11;14) were normalized by concurrently quantifying copies of the internal reference gene albumin for each dilution point.

Results. The sensitivity of such Q-PCR strategy, assessed by evaluating the rearranged gene copy numbers on positive sample curves, i.e. DNA from t(14;18)- and t(11;14)- positive cell lines serially diluted in 500 ng of healthy DNA, was of 8.5 rearranged gene copies/500 ng DNA (lower limit). Performance of standard curves was evaluated by collecting data from different (daily-prepared) curves (pFCL=24) and (pMCL=10), utilized over 1 year. Analysis of coefficient of variation (CV) calculated on the slope values from daily standard curves, displayed a high intra-assay (CVs 1,7% for pFCL and 1,9% for pMCL) and inter-assay (CVs 7,7% for pFCL and 5,9% for pMCL) reproducibility. As opposed to genomic DNA curves, both pFCL and pMCL showed a high stability even following long-term storage (> 1 year). To improve reagent resistance against unspecific DNase cleavage, we compared the performance of linearized and non-linearized forms of both pFCL and pMCL. The means of Ct values for both forms of plasmids did not show any statistically significant difference (Fisher’s Exact Test). Upon routine use in the context of clinical trials, our methodology was able to efficiently monitor MRD with a detection limit as low as 10 tumor cells/100000 normal cells. Conclusions. The described system: i) can be used to build robust standard curves for absolute quantification of any DNA target of interest; ii) displays a high intra-/inter-assay sensitivity and reproducibility; iii) is suitable for use with any Q-PCR strategy (TaqMan; SYBR Green)

P 084

A Novel Thermostable DNA Polymerase for Real-Time Quantitative PCR.

Nery J., Andersen M., Padmabandu G., Bishop J., Lee J., Mi Q., Finn P., Meredith R.

Invitrogen, United States of America

Email: jonathan.nery@invitrogen.com

Since the invention of Polymerase Chain Reaction (PCR), the technology has been routinely used in research and diagnostic laboratories across the world. The technology has seen many advances in reagent development where novel DNA polymerase formulations have contributed to the overall improved performance of quantitative PCR systems. The development of hot-start mechanisms to inhibit polymerase activity at reaction set up temperatures has simplified the workflow for end users and also improved PCR performance by limiting PCR artifacts.

Here we present a novel hot-start DNA polymerase formulation for use in quantitative PCR applications. The formulation is based on Thermus filiformis DNA polymerase, a thermostable DNA polymerase developed to perform better than Taq DNA polymerase. A novel antibody mediated hot-start mechanism is also described.

P 085

Comparison of different real time PCR SYBR® Green kits.

C Mazurier, CHAPEL A., A Elselmi

INSERM U832 Paris, EBI Cergy PontoiseFrance

Email: a.elmselmi@ebi-edu.com

Technical advances in the field of molecular biology, particularly for real-time PCR (RT-PCR), have greatly expanded the number of users, so that RT-PCR plays a crucial rule in numerous studies. A wide range of detection systems are available for RT-PCR, all based on the propagation of a fluorescent signal which is proportional to the initial amount of nucleic acid. Here we focus on the use of non-specific fluorescent dye, SPBR Green. SYBR green chemistry has proven to be the easiest and most cost-effective method employed for RT-PCR. Many companies have therefore developed a wide range of ready-made kits in order to facilitate rapid and reproducible results. As these kits are all ostensibly based on the basic chemistry our goal was to compare 8 different kits (kindly provided by ABgene, Applied Biosystem, Eurogentech, Finnezyme, Invitrogen, Qiagen, Sigma Takkara). in order to check if these different mixes have impact on different critical points as RT - PCR efficiency, sensitivity, specificity. To do this, eGFP gene was amplified with the same primers set for all kits. No obvious difference was observed.

P 086

Development of an external standard for RT-qPCR.

Françoise WESSNER, Véronique MONNET

INRA, Jouy en Josas, France

Email: francoise.wessner@jouy.inra.fr

The RT-qPCR is a very sensitive method but its reliability strongly depends on an appropriate normalisation. In order to avoid the bias caused by the fluctuation of an housekeeping gene, and because the use of several housekeeping genes (Vandesompele et al., 2002) is laborious and expensive, we have chosen to develop an external standard added to the RNAs before the retrotranscription step.

The aim of this study is the setting up of an external standard and its validation for bacterial gene expression measurement by RT-qPCR. Our model concerns the expression of oligopeptide binding proteins encoding genes during growth in milk. The oligopeptide transport system of Streptococcus thermophilus is fundamental for its optimal growth (Garault et al., 2002). It belongs to the ABC transporters family and is composed of 3 oligopeptide binding proteins (in our strain) encoded by the amiA1 _amiA2_ and amiA3 genes, 2 transmembrane proteins forming a channel and 2 ATPase providing energy to the system. Since the sequences of the amiA genes are very homologous, we increased the qPCR specificity with the TaqMan technology to bypass non specific amplification.

We choose a mRNA encoding the luciferase gene as external standard as it is absent in the lactic acid bacteria strains and commercialised by Promega. Its use allows the detection of DNA polymerase inhibitors. The use of this tool requires a very precise quantification of total RNA. We decided to do it with the Nanodrop since there is no need to dilute the sample. The quality of our RNA samples was checked with Agilent analysis ( Nano LabChip kit).

We have shown that the addition of 8.103 to 2.107 molecules of the luciferase mRNA/µg total ARN does not interfere with its own retrotranscription nor with the retrotranscription of target genes. For the rest of the study we added 106 molecules per sample.

The study of the amiA genes expression shows similar transcription profiles with an optimum expression in the middle of the exponential phase. The amiA3 gene is significantly more expressed than the amiA1 and amiA2 . These results - and the fact that the growth of a amiA3 mutant stop at an early stage (data not shown) - showed that this protein has a determinant role on bacterial growth in milk. It is important to note that we did not observe any variance in RNA quantity in our samples.

We demonstrated that RT-qPCR using luciferase standard is effective for absolute quantification in bacteria in absence of suitable housekeeping genes.

P 087

High-throughput selection and validation of qPCR reference genes for barley.

Skov Jakob, Hagedorn Peter, Lyngkjær Michael F.

Biosystems Department, Risø National Laboratory, Technical University of Denmark, DK-4000 Roskilde

Email: jakob.skov@risoe.dk

Due to sample-specific technical variability, transcript levels measured by qPCR need to be normalised to reference genes. However, many traditionally used reference genes have proven to be differentially regulated under some conditions, so efficient methods to select new stable reference genes are needed.

Using available datasets from the Affymetrix Barley 1 GeneChip we identified 38 stably expressed barley genes from the ~20.000 genes present on the microarray. We selected nine top ranking genes together with five traditionally used reference genes for testing in a large qPCR experiment. This experiment included leaves from three different barley genotypes, ± treatment with the pathogenic barley powdery mildew fungus and sampling at four different time points after infection. The genes were subsequently ranked according to stability using the geNorm software. Different sets of optimal reference genes, consisting of both new and traditional reference genes, were identified for each genotype. Only one gene, encoding an NADH dehydrogenase, was among the top four most stable genes in all three genotypes. Other genes among the top four in one genotype were unstable in other genotypes. A second qPCR experiment which included five biological replica, revealed three traditional and one novel reference gene to be significantly regulated (p<0.001) when comparing inoculated and un-inoculated control samples. Normalising to these genes would lead to erroneous conclusions. Finally three qPCR reference genes are recommended for experiment dealing with powdery mildew in the three genotypes respectively.

By proof of contradiction we conclude that presently one cannot assume that universally applicable reference genes exist for different genotypes even within a species. We speculate that such genes may not exist at all, and recommend that reference genes are (1) always validated for the specific setup in which they are to be used and (2) selected from a set of potential reference genes generated by analysis of microarray data. The presented work describes a method to producing such a list and a setup for validation.

P 088

Can real-time NASBA compete with qPCR for GMO detection?

Dany Morisset, Kristina Gruden

National Institute of Biology, Slovenia

Email: dany.morisset@nib.si


We developed a non-PCR based method for the multiplex quantitative detection of GMOs. This method called NASBA (Nucleic Acid Sequenced-Based Amplification) is a sensitive, isothermal, transcription-based amplification system for the specific replication of nucleic acid in-vitro. It is widely used for RNA target amplification in virus and bacteria detection. In this work, we show how this system can be successfully adapted for mono- and multiplex DNA-based detection of GMOs in a real-time fashion.

Materials and methods

Test samples and DNA purification

Genomic DNA was purified from plant material and reference flour samples containing defined percentages of GMO material. Concentrations of DNA were quantified by qPCR.

Multiplex DNA NASBA (conventional and real-time)

In a first amplification step, “tailed” primers were used in a single cycle Taq polymerase amplification to get the appropriate template for NASBA bordered by “universal” regions. Universal primers corresponding to the universal borders in the template DNA were used for NASBA amplification based on the NucliSens® Basic Kit (bioMérieux bv, Boxtel, The Netherlands). In the case of real-time monitoring, molecular beacons (MWG-BIOTECH AG Ebersberg) corresponding to specific sequences of the target DNA were added to the reaction mix. Otherwise, amplification of NASBA products was quantified using classical qPCR with specific probe for the target DNA.


The usual protocol for NASBA as proposed by the manufacturer failed in amplifying GMOs targets. The optimisation of the procedure included the addition of a first amplification step to generate a suitable template prior to the NASBA reaction itself. Another improvement was the use of “tailed” primers during this first step. In the following NASBA reaction, the use of “universal” primers avoided the problem of complex primers mix and allowed the multiplexing of the procedure. Several target elements from various GMOs were amplified in simplex NASBA reactions showing strong amplification when compared to the control DNA. The improved real-time and conventional NASBA was shown to be sensitive and specific.

Multiplex NASBA reactions were also performed aiming specie-, construct-, and event-specific target elements. Same amplification rates were found for each target element compared to simplex NASBA reaction. These results demonstrate the specificity of the first amplification step, and the non-discriminatory property of the “universal” primer set for any target element. Real-time and conventional NASBA showed the same results for multiplex amplifications. All results were compared to qPCR amplification parameters (efficiency, specificity, sensitivity…)


We show that due to its sensitivity, specificity, amplification efficiency and multiplexing, real-time NASBA can be used for DNA amplification in the goal of GMO detection. This method shows great potential to challenge with qPCR-based GMO detection.

P 089

Identification of heterozygous and homozygous sequence changes in the MUTYH gene by High Resolution Melting Analysis.

Rossella Tricarico1, Benedetta Ciambotti1, Roberta Sestini1, Maurizio Genuardi1 Claudio Orlando2.

1Medical Genetic Unit and and 2Clinical Biochemistry Unit Department of Clinical Physiopathology, University of Florence, Italy

Email: c.orlando@dfc.unifi.it

High-resolution melting analysis (HRMA) is a mutation detection method based on the principle that the melting curves of DNA fragments vary depending on base composition. Heterozygous samples can be detected with high sensitivity, whereas homozygosity for a nucleotide change (A to T or C to G transition) may not lead to significant curve shape or melting temperature changes compared to homozygous wild-type samples. Therefore, HRMA has been mainly applied so far to the detection of mutations associated with autosomal dominant or X-linked disorders. In this study, we have evaluated whether HRMA could be used for the identification of homozygous mutations in the MUTYH gene, implicated in the autosomal recessive form of intestinal polyposis (MUTYH-associated polyposis; MAP). HRMA was first tested on a set of 30 samples of known genotype at exons 7 and 13, where the mutations, 495a>g (Y165C) and 1145g>a (G382D) are located. These account for about 70% of pathogenic MUTYH äallelic variants among Caucasians. HRMA was conducted on a Rotor Gene 6000 Instrument (Corbett Research, Sydney, Australia). In order to generate a condition of artificial heterozygosity, test samples were mixed with a homozygous wild-type reference control sample in 85:15 proportions and amplified by PCR. Following PCR, amplicons were denaturated at 95°C for 1 min and then rapidly cooled to 40°C for 1 min to facilitate heteroduplex formation. Fluorescence difference plots were then generated. All samples heterozygous or homozygous for the 495a>g and 1145g>a mutations, as well as for other exon 7 and 13 sequence variants, were reliably identified by HRMA. The experiments were then repeated without mixing the test samples with the reference control DNA. We found that homozygous mutants could be consistently distinguished from homozygous wild type samples also under these conditions. Finally, we applied HRMA as an initial mutation screening test in a series of 14 new samples of unknown genotype. The results obtained were then confirmed by DNA sequencing, indicating that HRMA is a sensitive and reliable method for the identification of mutations in the hotspot exons of the MUTYH gene.

The Authors wish to thank Diatech (Jesi, Italy) for the technical support.

P 090

Internal quality assessment scheme for real-time PCR applications.

Helene Polin 1, Martin Danzer 1, Reinhard Haunschmid 2, Katja Hofer 1, Brigitte Fiedler 1, Johannes Pröll 1, Christian Gabriel 1

1Red Cross Transfusion Service of Upper Austria, Linz, Austria and 2Federal Agency for Water Management, Mondsee, Austria

Email: helene.polin@blutz.o.redcross.or.at

Real-time PCR is a key procedure of molecular diagnostics in a clinical setting. The reliability of results generated in routine diagnostics must be ensured by continuous internal and external quality controls. In contrast to existing quality assessment schemes, we developed an internal procedure exactly to control the validity of generated data based on the qualification of (1) real-time instruments and (2) enzymes and (3) statistical process control (SPC) of routine diagnostic assays.

For the qualification of supplies, an in-house real-time PCR was introduced. MS Excel-based statistical analysis of the Cp data was generated with results automatically displayed in Shewart control charts. This quality assessment scheme was applied in our molecular diagnostic laboratory for 17 months to all real-time PCR supplies and selected real-time PCR assays. Whereas all 30 different lots of the LightCycler FastStart DNA Master Hybridization Probes kit enzyme showed no irregularities, one outlier out of the133 data sets generated from 16 different LightCycler instruments was identified. Further maintenance revealed an insufficient fluorimeter device responsible for the inappropriate result.

SPC applied in the daily routine testing for Hepatitis B virus (HBV) identified 1.5% of the amplification runs with control data beyond the action control limits (+3SD) displayed in Shewart control charts. Results generated within these runs were assigned as invalid. Therefore, this scheme for quality control of real-time PCR supplies and assays is easily applied and useful for every molecular diagnostic laboratory and supports in every-day quality management.

P 091

miniProbe: Highly Specific Homo-Labeled Probe Technology for Real-Time qPCR.

Xin X. and Mao F.

AlleLogic Biosciences Corporation, Hayward, California, US

Email: shanex@allelogic.com

AllGlo probes are novel fluorogenic probes for nucleic acid detections in real time quantitative PCR. Unlike conventional dual labeled probes such as TaqMan probes, an AllGlo probe has two identical reporter dyes that are capable of quenching each other and become de-quenched once the probe is cleaved. AllGlo probes do not employ a quencher and have two signal-generating reporter dyes per oligo; they tend to generate much higher fluorescence signals. Furthermore, the simple composition translates into low manufacturing cost.

Recently, we have developed the second generation of AllGlo probes, called miniProbes. The miniProbes are significantly shorter than the older version of AllGlo probes. They have a calculated Tm of 55-60 deg. C contributed solely by the nucleic acid sequence itself, and the length can be as short as 15-16 nucleotides. Although the calculated Tm is lower than the annealing/extension temperature used in the universal protocol, the miniProbe performs better than the regular AllGlo probe that has a calculated Tm of 65-70 deg. C when they are compared in TaqMan assays. miniProbes with shortened oligo length make quenching more efficient and boast low fluorescence baselines. The ∆Rn is at least equal or better than TaqMan BHQ probes or TaqMan MGB probes. We discovered that a miniProbe offers higher specificity than Taqman probe or TaqMan MGB probe in detecting single point mutations. Unlike TaqMan MGB probes, we have not found a single case that a miniProbe inhibits PCR reactions.

P 092

Normalisation of mRNA levels using expressed Alu repeats (EARs) to investigate immunoregulation.

Nina Witt1, Jo Vandesompele2, Alimuddin Zumla1, Graham Rook1, Jim Huggett1

1Centre for Infectious Diseases & International Health, University College London, UK and 2Centre for Medical Genetics, Ghent University Hospital, Belgium

Email: n.witt@ucl.ac.uk

The environmental Mycobacterium vaccae has been shown to induce regulatory T cells which inhibit the allergic response in mice. We are studying the immunoregulatory effects of M. vaccae on human dendritic cells. To analyze cytokine expression changes in dendritic cells after treatment with M. vaccae , we first have to perform data normalisation. Normalisation of quantitative RT-PCR data is of critical importance to control for experimental variation. Using the expression of multiple internal reference genes is currently the most accurate strategy to control for experimental error and identify true changes in transcription. However, identification and measurement of multiple reference genes can be time-consuming and expensive.

To tackle this problem, we are working on an alternative normalisation strategy: expressed Alu repeats (EARs) that has the potential to provide accurate normalisation without measuring multiple reference genes. Alu repeats are short interspersed nuclear elements (SINE) that comprise ~10% of the human genome and are frequently expressed in the untranslated regions of mRNAs. Primers designed to amplify the Alu consensus sequence are used, allowing the expression of many different transcripts to be measured at the same time. As there are so many EARs, they provide a good measure of cDNA and could be used to compensate for experimental variation. This would potentially allow similar accuracy to using multiple reference genes with a much simpler strategy.

The high frequency of the Alu sequences that provide an excellent normalisation strategy also cause a major problem as these assays are highly susceptible to contamination. Currently we are optimising this strategy and are particularly interested in the source of this contamination. This work will decide whether we will utilise EARs as our final normalisation strategy when measuring cytokine responses in dendritic cells. We will also discuss whether certain defined levels of contamination can ever be acceptable.

P 093

Quantitative Analyses of TACC1 Alternative Splicing Events in Gastric Cancer.

Silina K., Abols A., Kalnina Z., Line A.

Latvian Biomedicine Research and Study Centre, Latvia

Email: karina@biomed.lu.lv

Introduction . In order to look for novel cancer specific proteins that could be used as diagnostic/prognostic markers or new therapeutic targets in gastric cancer we applied SEREX technique to gastric cancer cDNA expression library. As a result we identified 14 antigens including the mitotic spindle binding protein TACC1. Its serum reactive clone represented a previously unknown splice variant and the immune response to it was exclusively cancer patient specific. We analyzed it further to find out the possible reasons of its immunogenicity and its possibilities of application in therapy.

Methods . We used 5`RLM-RACE approach to obtain full length mRNA sequence of TACC1 which was absent in the serum reactive clone. To analyze expression patterns of TACC1 in various normal as well as gastric cancer/adjacent normal tissue pairs we performed RT-PCR and qRT-PCR. For qPCR data normalization we used a normalization factor calculated from 3 most stable genes in a given tissue panel. We amplified 7 housekeeping genes that were published to be used for qPCR in similar panels – YWHAZ, TBP, PolR2A, TUB3A, GAPDH, ACTB, PGK1 and determined the 3 most stable genes by GeNorm – PolR2A, ACTB, TUB3A for various normal tissues; ACTB, YWHAZ, TBP for tumour/adjacent normal tissue panel.

Results . So far we have identified 17 transcripts of TACC1 which differ in exon composition as well as promoter usage. Each of these transcripts can be spliced with or without a novel exon (Ex8) that was present in SEREX clone. The RT-PCR analyses showed that TACC1 isoforms without Ex8 are expressed in most normal tissues, while Ex8 is being included predominantly in gastric cancer and normal brain. Our qPCR results showed that there is a distinct expression pattern for each splice variant and that splicing deregulation rather than changed transcription intensity causes overexpression of Ex8 containing isoforms in cancers comparing to adjacent normal tissues. To find the possible reasons of Ex8 inclusion in cancer we looked at mRNA expression levels of 4 splicing factors which are predicted to bind enhancer elements of Ex8. However we were unable to detect any differences between normal and cancerous tissues meaning that other splicing regulatory events are disturbed and result in Ex8 inclusion in gastric cancer transcripts. In several isoforms the inclusion of Ex8 disturbs a nuclear localization signal. Whether that might contribute to oncogenic events remains to be studied.

Conclusions . We have identified 17 different transcripts of TACC1 and shown that in about 50% of gastric cancer cases the alternative splicing of TACC1 is deregulated through mechanisms other than splicing enhancer transcription intensity. It is not known how it contributes to the immunogenicity of TACC1 nor how that implicates in cancerogenesis. Isoforms containing Ex8 can be used as gastric cancer biomarkers, but as they are present abundantly in the brain, their use as therapeutic targets is unlikely.

P 094

Real-time RT-PCR protocol adaptation case in instrument change.

Karus A., Karus V.

Estonian University of Life Science, Estonia

Email: avo.karus@emu.ee

For basis for adaptation in instrument was used the 2-step RT-PCR protocol for quantification of elongation factor EF-Tu optimised for Smartcycler. We used for transference Roche recommendations for optimisation as well as Roche LightCycler RNA master SYBR Green I method manual and we performed 1-step RT-PCR using same primers as for Smartcycler. Primers: Ef-Tu forward - GAGATGGAGAATACGTCTTCGA; Ef-Tu reverse - ACCAGAGCGTGCGATTG. All standards and samples were processed in duplicates for both assays. SYBR Green I was used for detection format. All changes were based only on theoretical basic knowledge. RT was performed at 61 C in 20 min. Annealing temperature was set according to calculated primer melting temperature on 50 centigrade. To ensure higher efficiency the denaturizing step was 5 s, annealing 8 and extension step 15 seconds (in optimised protocol for Smartcycler accordingly 15, 20 and 15 s). Temperature ramp rate was set to maximum except from annealing to extension only 2 C/s. The changes done in instrument and RT-PCR method change allowed getting quantification with excellent efficiency (1.96) and good uniformity. Results showed robustness of LightCycler system as well as reagents, that allowed obtain reliable quantitative results in low copy number (less than 100) of target on LightCycler 2.0 instrument with high specificity of amplification, checked by melting curve analysis in less than 80 minutes (equal to RT-step duration in 2-step PCR protocol) without any optimisation steps. The single amplification product has Tm 80.1 C.

P 095

Reference gene stability in hepatocyte primary cell cultures of Atlantic cod (Gadus morhua).

Liv I. R. Søfteland1 and Pål A. Olsvik1

1National Institute of Nutrition and Seafood Research, PO Box 2029 Nordnes, N-5817 Bergen, Norway

Email: lso@nifes.no

In salmonids hepatocyte primary cell cultures are commonly used for toxicity testing in bioassays. So far no one has described a protocol on how to use Atlantic cod hepatocytes in bioassays, mainly due to the high fat content in these cells, causing the cells to burst during isolation. In this work we describe an experiment in which we were able to isolate intact liver cells from one adult mature female individual (3.0 kg), expose them to three concentrations of PCB 138 (0.1 µM, 1 µM and 10 µM plus one unexposed control group, n=3) and extract high quality RNA (average RIN value 9.6±0.2) for gene expression analysis. PCB 138 is one of the most widespread PCB congeners, and of major concern in the marine environment (AMAP, 1998). This PCB congener has previously been used in in vitro exposure studies (Gooch et al., 1989; Schmitz et al., 1995). We were able to quantify the transcription levels of three reference genes and four target genes encoding proteins known to be affected by oxidative stress, namely glutathione peroxidase (GSH-Px), glutathione reductase (GR), glutathione S-transferase (GST) and Mn superoxide dismutase (Mn SOD), in addition to the detoxifying enzyme CYP1A and vitellogenin (Vgt). The results showed that heat shock cognate 70 (the non-inducible form of HSP70) was slightly more stable than elongation factor 1A and beta-actin, with M-values of 0.152, 0.165 and 0.171, respectively (analyzed by GeNorm) (Vandesompele et al., 2002). CYP1A is one of the genes known to be affected by PCB 138 (MacFarland & Clarke, 1989), but surprisingly, we were not able to quantify significant differences between the control and the exposed groups. Nor could we measure significant differences in transcription levels between the control and the exposed groups for Vtg, GSH-Px and GR. Further only minor, non-significant differences were found between the control group and the groups of cells exposed to PCB 138 for GST and Mn SOD.

1. AMAP (1998). AMAP Assessment Report: Arctic Pollution Issues. Arctic Monitoring and Assessment Programme (AMAP). Oslo, Norway.

2. Gooch, J.W., Elskus, A.A., Kloepper-Sams, P.J., Hahn, M.E. & Stegeman, J.J. (1989). Toxicology and Applied Pharmacology, 98, 422-433.

3. McFarland, V.A. & Clarke, J.U. (1989). Environmental Health Perspectives, 81, 225-239.

4. Schmitz, H.-J., Hagenmaier, A., Hagenmaier, H.-P., Bock, K.W. & Schrenk, D. (1995). Toxicology, 99, 47-54.

5. Vandesompele, J., Preter, K.D., Pattyn, F., Poppe, B., Roy, N.V., Paepe, A.D., Speleman, F. (2002). Genome Biology, 3 (7), Research0034.1-0034.11.

P 096

Reference genes for studying gene expression by Real-Time Quantitative RT-PCR in Arabidopsis thaliana exposed to toxic concentrations of Cadmium and Copper.

Tony Remans, Karen Smeets, Jaco Vangronsveld, Ann Cuypers

Environmental Biology, Hasselt University, Belgium

Email: tony.remans@uhasselt.be

From a general biological and a more specific physiological perspective, essential micro-elements (e.g. Copper, Cu) and non-essential trace elements (e.g. Cadmium, Cd) can be distinguished within the group of metals. From certain exposure levels, non-essential trace elements are toxic for plants, and even though micro-elements are essential for the plant, they can become toxic for plants at higher concentrations. This can cause a disruption of physiological processes, such as photosynthesis, transpiration,… which can lead to chlorosis, necrosis, reduced growth… Also at lower exposures to trace elements, when no obvious changes to the plant can be detected visually, a number of biological and molecular changes are taking place. Several studies have observed that the exposure of plants to toxic concentrations of these elements provokes oxidative stress. Oxidative stress occurs when the balance between oxidants (such as reactive oxygen species, ROS), and antioxidants (which include certain enzymes and metabolites), is shifted towards the oxidants.

In our studies of Arabidopsis thaliana exposed to Cd or Cu, we quantify gene expression of anti-oxidative defence genes (superoxide dismutases, catalases, peroxidases) and genes that can cause oxidative stress (NADPH oxidases, lipoxygenases). Especially when studying Cu stress, we found that some widely used reference genes for relative quantification were not stably expressed between different treatments. In order to determine more suitable reference genes, we tested a series of genes for Arabidopsis as proposed by Czechowski et al, and used the geNorm algorithm (Vandesompele et al) to identify the most stably expressed reference genes in roots and leaves of Arabidopsis under Cu and Cd stress.

P 097

Regulation of neuroglobin, cytoglobin and myoglobin in a hypoxia-tolerant mammal, the subterranean mole rat Spalax ehrenbergi.

Frank Gerlach1,2, Aaron Avivi3, Thorsten Burmester2, Eviatar Nevo3, Thomas Hankeln1

1Institute of Molecular Genetics, University of Mainz, 55099 Mainz, Germany, 2Biocenter Grindel, Animal Physiology, University of Hamburg, 20146 Hamburg, Germany and 3Institute of Evolution, University of Haifa, Haifa 31905, Israel

Email: frank.gerlach@uni-hamburg.de

Neuroglobin, primarily expressed in neurons of the CNS and PNS, and cytoglobin, found in fibroblasts of all organs and in distinct neuronal cells, are recently discovered O2-binding respiratory proteins of vertebrates. Their physiological function is discussed related to O2 supply, ROS scavenging, NO detoxification and other mechanisms.

Here we report expression patterns of these globins and of myoglobin in mole rats which are able to survive extended periods of severe hypoxia in their underground burrows without damage. Quantitative RT-PCR and Western blot analyses show that both globins are expressed at elevated levels in Spalax versus rat. This suggests that both globins contribute to a pre-adaptation of Spalax towards a lack of O2. After extended periods of hypoxia (10% O2 for 22/44hrs), both globins are down-regulated at the mRNA level in both species. The largely parallel pattern of gene regulation of neuroglobin and myoglobin suggests similar cellular functions.

Cytoglobin shows an augmented normoxic expression in Spalax vs. rat only in brain tissue, but not in heart and liver. Hypoxia specifically induces cytoglobin in heart and liver of both mammals, with the most pronounced response (12x up) in Spalax heart. That indicates that cytoglobin possibly fulfils different roles in fibroblast-like cells and in neurons.

P 098

Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies.

Spinsanti G.1, Panti C.1, Casini S.2, Marsili L.2, Fossi M.C.2

1Department of Evolutionary Biology, University of Siena, Italy and 2Department of Environmental Sciences, University of Siena, Italy

Email: spinsanti@unisi.it

Odontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants with endocrine disrupting capacity. Skin biopsies of marine mammals can be easily obtained from free-ranging specimens using non-lethal techniques and previous ecotoxicological studies have assessed their suitability as fresh biological material for the determination of toxicological hazard. Up to now, quantitative Real-Time PCR has never been used in skin biopsies to quantify the expression of target genes induced by exposure of animals to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression levels. In this context, a systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers. Ten commonly used house-keeping genes were partially sequenced in the striped dolphin ( Stenella coeruleoalba ) and, for each gene, PCR primer pairs were specifically designed and tested in qRT-PCR assays. The expression of these potential control genes was examined in 30 striped dolphin skin biopsy samples, obtained from specimens inhabiting the north-western Mediterranean Sea. The stability of selected control genes was determined using three different VBA applets: geNorm , NormFinder and BestKeeper . Glyceraldehyde-3P-dehydrogenase (GAPDH) and tyrosine-3-monooxygenase (YWHAZ) always rank as the two most stably expressed HKGs according to the analysis with geNorm and NormFinder, and are defined as optimal control genes by BestKeeper. Ribosomal proteins L4 (RPL4) and S18 (RPS18) also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC), phosphoglycerate kinase1 (PGK1), hypoxanthine ribosyltransferase (HPRT1) and β-2-microglobin (B2M) show variable expression levels among the studied samples and appear as less suitable reference genes. The three VBA applets tested in this work, based on different analytical procedures, produce highly comparable results. The genes encoding for YWHAZ and GAPDH have the most stable expression patterns and appear as highly reliable controls. Potentially useful reference genes are also those encoding for the ribosomal proteins L4 and S18. This work provides background essential information for studying expression patterns of several potential genes of interest as biomarkers of exposure to contaminants of free-ranging marine mammals and constitutes the first effort towards the routine application of qRT-PCR for the evaluation of toxicological hazard in cetacean species.

P 099

The expression of plasminogen activator (PA) and their inhibitors during different functional stages in the bovine ovary.

Berisha Bajram 1, Kliem Heike 1, Welter Harald 1, Pfaffl W. Michael 1, Pfarrer Christiane 2, Schams Dieter1

1Physiologie Weihenstephan, TUM, Germany and 2Dept. of Obstetrics and Gynecology, Justus-Liebig-University, Giessen, Germany

Email: berisha@wzw.tum.de

The PA system members, tissue plasminogen activator (tPA), urokinase PA (uPA) and their inhibitors (PAI-1 and PAI-2), are supposed to be important regulators of the tissue remodeling in the ovary. The aim of this study was to evaluate the mRNA patterns of tPA, uPA its receptor uPAR, PAI-1 and PAI-2 during different functional stages in the bovine ovary. In our first experiment ovaries containing preovulatory follicles or new CL were collected by transvaginal ovariectomy at 0, 4, 10, 20 and 25h (follicles) and 60h (CL day 1-2) relative to injection of GnRH. Second experiment; corpora lutea were divided in following groups: Days 1-2, 3-4, 5-7 and 8-12 of the estrous cycle. Third experiment; cows in the mid-luteal phase (days 8-12) were injected with Cloprostenol (Estrumate) for induction of luteolysis and CL were collected at 0, 0.5, 2, 4, 12, 24, 48 and 64h after injection. Real-time RT-PCR (Rotor Gene) was employed to determine mRNA expressions. Experiment 1: tPA mRNA increased 4h after GnRH (during LH surge) and remained high during the whole experimental period. uPA transcripts did not change in follicle classes during periovulation but increased significantly only after ovulation. Both uPAR and PAI-1 mRNA expression increased in follicle group at 4h after GnRH, in order to increase again after ovulation. Experiment 2: uPA mRNA increased on days 8-12 of estrous cycle. In contrast, PAI-1 und PAI-2 mRNA were high on days 1-7 and decreased significantly on days 8-12 while uPAR and tPA mRNA did not change throughout the investigated periods. Experiment 3: After induced luteolysis the PA system members (tPA, uPA, uPAR) and their inhibitors were upregulated from 2h till 64h, only tPA increased already after 0.5h. The strong upregulation of tPA, PAI-1 and uPAR during LH surge suggest them to be important mediators of LH dependent rupture of bovine follicle. In addition these data suggest, that members of the PA system play an important role during CL formation and function as well as in degradation of extracellular matrix during induced luteolysis in cow. Supported by DFG (BE 3189/2-1).

P 100

The Intricacies of Multiplexing Revealed through a Pathogen Detection Assay.

V. Evan Messenger, Ben Sowers

Biosearch Technologies, United States of America

Email: evan@biosearchtech.com

The intensifying demand to detect pathogens in food products, agriculture, and the environment can be met with speed and confidence by multiplexing PCR assays together. Spectrally-distinct fluorophores and quenchers provide the ability to detect multiple genetic signatures that distinguish closely related strains, all within the same reaction chamber. Here we present amplifications from a TaqMan assay engineered to detect the virulence factors of Bacillus anthracis and identify several common laboratory strains, while also distinguishing a closely-related organism. Competing amplifications place new demands on the performance of each assay, and we explore the added effort required to successfully multiplex sequence design, master mix formulation, and performance optimization. With pathogen detection as one possible application, we show that multiplexing is ideally suited toward assays that will be run regularly, or where sample material may be precious.

P 101


Ann-Elise Olderbakk Jordal1, Pål. A. Olsvik2, Tom Ole Nilsen1, Jon Amberg3, Camilla Myr1, Mali Bjerkhaug, Yuko Kamisaka1, Ivar Rønnestad1.

1Dept of Biology, University of Bergen, N- 5007 Bergen, Norway, 2National Institute of Nutrition and Seafood Research (NIFES), Post box 2029 Nordnes, 5817 Bergen, Norway, 3Aquaculture Research Institute, University of Idaho, Moscow, ID 83844, U.S

Email: ann-elise.jordal@bio.uib.no

Housekeeping genes may vary in their expression with the experimental conditions. When studying ontogeny of genes that are influenced by both diet and hormonal stimuli, it is important to evaluate the use of housekeeping genes for normalisation. The growth rate of the developing larvae also challenges the use of housekeeping genes as cells divide at a high rate. Our studies aim to describe the ontogeny of the digestive system in developing marine fish larvae. This information is vital in order to formulate proper feeds in aquaculture. Similar to most other marine fish larvae, Atlantic cod has a very simple digestive system at the onset of exogenous feeding and may therefore have a limited capacity to completely digest complex proteins into free amino acids (1). Absorption of peptides via the oligopeptide transporter (PepT1) might therefore be very important to utilise dietary proteins (1,2). However no knowledge exists on the spatial and temporal expression of PepT1 and how development and diet influence the expression of PepT1. Digestion is a complex, but closely orchestrated process, involving enzyme and fluid secretions and motility, culminating in absorption and evacuation. CCK plays a key role in the stimulation of pancreatic enzyme secretion, gallbladder contraction, intestinal peristalsis, delaying of gastric emptying and control of food intake (3). CCK expression is therefore believed to increase in a temporal fashion (4). Our aim is to characterise the ontogeny of the digestive system and examine gene expression of PepT1 and CCK in Atlantic cod, Gadus morhua L, by examining the temporal and spatial expression of PepT1 and CCK using in situ hybridisation and q-PCR. Other strategies for normalisation will be evaluated. The functional implications for the processing capacity of the larval digestive tract will be discussed.

P 102

Validation of the Plexor™ Primer Design System.

Katharine Hoffmann, Benjamin Krenke, Cynthia Sprecher, and Douglas Storts

Promega Corporation, United States of America

Email: doug.storts@promega.com

The Plexor™ Primer Design System was used to design primers for twenty different human mRNA targets suggested by a collaborator. Each primer pair was designed to span an intron, minimizing the potential of generating an amplification product from genomic DNA. A BLAST search was performed against the NCBI database to verify the primers were specific for the mRNA target of interest. All primer pairs were designed for use in duplex reactions targeting a ‘housekeeping’ gene (GAPDH). Quantitative RT-PCR was performed with the Plexor™ Two-Step qRT-PCR System. We will present data demonstrating the results of the validation study.

P 103

Validation of two reference genes for mRNA level studies of murine disease models in neurobiology.

Meldgaard M.

Medical Biotechnological Center, University of S Denmark, Denmark

Email: mmeldgaard@health.sdu.dk

When quantifying relative mRNA level changes using RT rt-PCR, for a given set of experimental conditions or a given disease model, identification of an unaffected and unchanged reference gene is necessary. The reference gene enables normalization of test gene data to ensure their reliablility. Among the factors that may compromise the reliability of experimental data obtained in RT rt-PCR and make normalization against reference gene(s) necessary are degradation of the sample RNA, and the presence in the extracted RNA of reverse transcriptase (RT) inhibitors.

We present data from evaluation and validation of the genes encoding hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1) and glyceraldehyde phosphate dehydrogenase (GAPDH) as individual reference genes in RT rt-PCR based mRNA level studies involving four murine neurological disease models. We found relatively small variations of HPRT1 and GAPDH mRNA levels, changes that may even be accounted for by the intrinsic variability of the combined RT rt-PCR processes. We conclude both genes are suitable as reference gene with these four models, provided quantification of subtle changes is avoided. We generally consider only changes in relative mRNA levels exceeding 2 fold for reproducible and reliable using only one reference gene.

We furthermore found, that given certain experimental conditions, normalization to total RNA alone, i.e. without normalization to a reference gene, provides for equally reliable relative mRNA level results. The required experimental conditions encompass RNA extraction without RNA degradation, absence of RT inhibiting impurities in the RNA extracts, precise quantification of RNA concentrations, and precise handling and pipetting throughout the RT and rt-PCR preparations.


Meldgaard M, Fenger C, Lambertsen KL, Pedersen MD, Ladeby R, Finsen B (2006) Validation of two reference genes for mRNA level studies of murine disease models in neurobiology. J Neurosci Meth 156: 101-110.

P 104

Use of Synthetic Template Oligonucleotides for Absolute Quantification of Transcripts in Canine Osteoarthritis.

Melissa Mariani, Philip Day

University of Dortmund, Germany

Email: philip.j.day@manchester.ac.uk

Real-time reverse transcriptase quantitative polymerase chain reaction (real-time RT-qPCR) is used for the precise measurement of gene expression in biological processes. For sample comparison in the detection of altered gene expression through amplification by using real-time RT-qPCR, the need for normalisation is required. Reference genes, also known as housekeeping genes, are assumed to have consistent expression across all samples under investigation, which enables them to be used to normalize the quantity of target genes. Currently, no universal reference gene exists that can be used across all tissue types in a single organism. Therefore, it is of great importance to select the best fit reference genes for each sample. Absolute quantification uses a standard curve of known concentrations of the RT qPCR amplicon sequence under investigation to determine the total mRNA of each transcript. Through a series of calculations one can determine the number of mRNA copies present per unit quantity of total RNA. The target gene expression values are normalized to the average reference gene expression value giving the number of target gene copies per molecule of reference gene. Unlike absolute quantification, relative quantification uses cycle threshold (Ct) values and normalizes each sample target gene expression to the average Ct value of the applied reference genes for each sample. In this experiment, microarray data was used to identify reference genes (CG14980-PB, IMP and MRPS7) produced from total RNA isolation in canine stimulated and unstimulated ligamentocytes and monocytes. RT qPCR assays were then designed for these reference genes and the target gene (IL-6) and applied to the cell samples. Synthetic template oligonucleotides (TOs) were designed for each of the genes of interest. Absolute and relative quantification methods were carried out to compare gene expression. The results showed analogous rank order between using relative and absolute quantification methods. IL-6 expression tended to be slightly higher using the relative quantification method compared to absolute quantification. However, standard error values were large enough that significant changes in expression were unlikely. In conclusion, although absolute quantification shows similar results in gene expression analysis to relative quantification, it has less error. By calculating the number of copies of molecules of gene transcripts, it produces a more robust value compared to analyzing the cycle threshold for relative quantification. Therefore, the use of synthetic TOs for normalizing gene expression via absolute quantification should be investigated further.

Additional posters:


Detection of defective PCR samples with module Outlier of Kineret software.

Bar T., Tichopad A. and Dahan E.

Labonnet, Israel

Email: tzachi.bar@labonnet.com

For proper quantification with real-time PCR, compared samples should have similar kinetics. Outlier is a computational tool implemented within Kineret software that analyses real-time PCR data and verifies the fulfilment of this prerequisite by detecting PCR samples with defective kinetics. By disabling such samples from the analysis, Outlier improves the overall results.


To identify defective PCR:

1.Characterize the kinetics of each amplification curve by several parameters.

2.Define a reference set consisting of well performing samples, usually the standard curve samples.

3. Self-asses the reference set for PCRs with outlying kinetics.

4.Test the kinetics of each unknown PCR against the reference set.

5. Use replicates at a given experimental level (e.g. PCR replicates, RT replicates) to detect PCRs with outlying CT.

6. Intersect the results of kinetics outlier detection and CT outlier detection.


Allergen determination in food by multiplex qPCR.

Veronika Dvorak, Franziska Zimmerli, Alda Breitenmoser, René Köppel

Email: rene.koeppel@klzh.ch

Official Food Control Authority of the Canton of Zürich Two to six percent of the human population exhibit allergic reactions due to the consumption of food containing allergens. In order to increase the well being and safety of these people, food samples are regularly examined for the presence of allergens and the compliance of declaration. Currently, there are 18 allergens listed in the food law of EU and Switzerland. In principle, food samples need to be analysed for all these allergens. Although only few food samples have to be tested for all possible combinations, still these analyses are very laborious and time-consuming. Therefore, multiplex PCR is a promising approach to produce the results in a more economic and fast way. Here, we present two tetraplex qPCR-systems. They simultaneously measure contents of peanut (Ara-h2-gen), hazelnut (Cor-a-1-gen), celery (Mannitol-dehydrogenasegen), soy (Lectin-gen), egg (TGF-â3-gen, milk (Cyt-B-gen, almond (Pru-A1-gen) and sesame (Oleasin-gen), respectively. DNA of the following organisms were isolated and tested for possible crossreactions in both multiplex PCR systems (AllAll A and AllAll B): 8 nuts (almonds, walnut, macadamia, cashew, pistachio, pecan, brazil nut, coconut) 10 legumes (white beans, lentils, kidney beans, mung beans, kidney beans, chickpeas, peas, runner beans, lupinus albus and lupinus august) 7 animals and their products (beef, chicken, eggpowder, whole egg-powder, pig, lamb, turkey, goat) 11 herbs (parsley, chive, nutmeg, onion, garlic, white pepper, cinnamon, aniseed, cloves, paprika, sesame) 6 fruits and vegetables (carrot, tomato, potato, apricots, peach, plum) rice and wheat. The only cross-reactivity above 1% emerged using peach and apricot as a template. These lead to a 10% signal in the hazelnut system and in case of apricot to a 1.2% signal in the almond-system. These two tests exhibit a good accuracy and precision (see Table 1 and 2) in the range of 0.01% to 10%. In order to check for the applicability of these PCRsystems forty bakery products (e.g. cookies, cereals, mueslibars, chocolate chips) were analysed in parallel by ELISA and by PCR for the presence of almond and hazelnut. The comparison of these results revealed a qualitative accordance of 97% for hazelnut and 98% for almond. These two multiplex PCR-systems proved their applicability for routine analysis.


Biomarker Discovery Using Arrays Printed With the BioOdyssey™ Calligrapher™ MiniArrayer.

J. A. Wibbenmeyer,1 N. Navarro,2 P. E. Schwartz,2 A. VanMeter,3 V. S. Calvert,3 J. D. Wulfkuhle,3 E. F. Petricoin III,3 L. A. Liotta,3 V. Espina,3

1 Gene Expression Division, Bio-Rad Laboratories, Hercules, CA, USA 2 Protein Biotechnologies, Inc., Ramona, CA;  3 Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, US

Email: teresa_rubio@bio-rad.com

The use of microarrays in transcriptional profiling has provided scientists with a wealth of information. This same technology has the potential to accelerate biomarker discovery in cancer research through the examination of protein expression and posttranslational modifications in cells, tissues, or serum in a multiplexed miniature environment. Reverse-phase protein arrays (RPPAs) are prepared from tissue or cell lysates from multiple samples that are arrayed on a slide and then probed with specific antibodies. These arrays are being used to uncover biomarkers in ovarian (Wulfkuhle et al. 2003) and breast (Cowherd et al. 2004) cancers, as well as in leukemia (Tibes et al. 2006). In this poster, we demonstrate the preparation of RRPAs for biomarker discovery by the BioOdyssey Calligrapher miniarrayer.


Novel Uses of Microarrays in Detecting Gene Silencing.

Elizabeth T. Jordan, Cathleen Karlak, Teresa Rubio, Luis Ugozzoli, and Jamie Wibbenmeyer.

Gene Expression Division, Bio-Rad Laboratories, Hercules CA 94547, US

Email: teresa_rubio@bio-rad.com

DNA microarrays allow many simultaneous parallel measurements and transcriptional profiling using these arrays has provided scientists with a wealth of information. The same technology can be used to build protein arrays, which hold similar promise. We describe here an approach using protein arrays to screen cells for gene knockdown by b-actin siRNA and to examine changes in levels of the actin-binding proteins destrin and cofilin. Arrays were produced on the benchtop with a BioOdyssey™ Calligrapher™ miniarrayer. Antibodies against b-actin and phosphorylated cofilin (p-cofilin) were tested for specificity using western blots. In one experiment, arrays were processed to monitor the concentration of b-actin in cells, with a standard curve of purified human actin printed on the grids. In another experiment on the same slide, changes in phosphorylation levels of cofilin were detected with an antibody specific for p-cofilin. We demonstrate that printed protein arrays can be screened using antibodies either singly or in pairs. Finally, the microarray results were validated using qPCR.


β-Actin Gene Silencing via siRNA and Its Effects on Protein Profiles.

Todd Yeck, Katrina Academia, Teresa Rubio, Ning Liu, Tim Wehr, Steve Freeby, Yuan Yan, Joseph Terefe, Keith Hamby, and Aran Paulus.

Gene Expression Division, Bio-Rad Laboratories, Hercules, CA, USA

Email: teresa_rubio@bio-rad.com

RNA interference (RNAi) is a powerful tool used to modulate gene expression and determine gene function. Delivery of small interfering RNA (siRNA) into cells can result in degradation of a targeted messenger RNA (mRNA) and reduction of its protein product. Resulting changes in mRNA levels can be effectively monitored by RT-qPCR, while two-dimensional gel electrophoresis (2-DGE) allows the monitoring of changes in the expression levels of proteins associated with the function of that gene in a cellular pathway. Actin filaments are major cytoskeletal structures that play important roles in many cell physiological behaviors, such as migration, proliferation, and differentiation. The proper function of actin is dependent on the highly dynamic assembly and disassembly of its filaments. Many proteins interact with actin to regulate the cytoplasm through crosslinking, bundling, capping, or severing of actin filaments (Weeds 1982). In this study, we examine changes in the protein profiles of HeLa cells after siRNA-mediated knockdown of β-actin. Any changes in protein expression resulting from β-actin knockdown may be directly or indirectly associated with actin filament function.


Parameters of effective siRNA transfection using siLentFect Lipid reagent.

Teresa Rubio, Joseph Terefe, Melody Yang, Michael Sturges, Steve Kulisch and Keith Hamby

Gene Expression Division, Bio-Rad Laboratories, Hercules, CA, USA

Email: teresa_rubio@bio-rad.com

Any changes ???   =>   please contact the scientific organizer Michael W. Pfaffl via    qPCR2007@wzw.tum.de