qPCR 2007 Symposium TALK Presentations

Abstracts - talk presentations

Advances in Quantitative PCR for Research and Diagnostic Applications.

Thomas W. Myers

Program in Core Research, Roche Molecular Systems, Alameda, CA 94501, US

Email: thomas.myers@roche.com

We have come a long way in developing the processes of reverse transcription and/or DNA amplification of RNA and DNA targets since the introduction of PCR. Many new technologies have been developed; from carryover contamination control, single enzyme RT/PCR, HotStart methodologies, and numerous strategies for quantitative PCR. Of particular interest for the detection of nucleic acid targets requiring high sensitivity and high specificity, especially for quantitative PCR strategies, the development of HotStart techniques ranging from barrier methods (wax), antibodies directed against DNA polymerases, reversible chemically modified DNA polymerases, and more recently chemically modified oligonucleotide primers, has allowed for enhanced ease of use, reaction robustness, and improved reproducibility. Similar to the advancement in the enzymatic and chemical aspects of these reactions, there has been a continual evolution in the instrumentation available for thermocycling and detection. An ever increasing toolbox of reagents and technologies has been contrived to meet the ever increasing and diverse needs and demands of the scientific and medical communities for both basic research and diagnostic applications. A historical reflection of these developments, current approaches, and future enhancements will be presented.

Main Session:   microRNA – siRNA Applications

Chair:   V. Benes / G. Shipley

Lecture hall   HS 14

Functional analysis of microRNA-containing protein complexes in human cells.

Meister G.

Max Planck Institute of Biochemistry, Germany

Email: meister@biochem.mpg.de

Small regulatory RNAs such as short interfering RNAs (siRNAs) or microRNAs (miRNAs) have been discovered in the past and it is becoming more and more apparent that these small molecules have key-regulatory functions. Such small RNAs are found in all higher eukaryotes and play important roles in cellular processes as diverse as cell differentiation, hormone secretion or stress response. SiRNAs guide sequence-specific cleavage of perfectly complementary target RNAs, whereas miRNAs associate with partially complementary target mRNAs and repress their translation. It is becoming more and more apparent that miRNAs can also influence the stability of mRNAs by guiding de-adenylation followed by mRNA decay. Interestingly, miRNA expression has also been associated with different diseases including neurodegenerative disorders as well as cancer. A detailed investigation and an accurate quantification of miRNAs in different tissues will therefore provide a better understanding of the molecular basis of such diseases and may ultimately lead to novel means of diagnosis.

MicroRNA profiling toolbox: points to consider.

Vladimir Benes, Mirco Castoldi, Sabine Schmidt, Martina Muckenthaler

EMBL, EMBL-University of Heidelberg Molecular Medicine Partnership Unit

Email: benes@embl.de

MicroRNAs (miRNAs) are now recognized as an important class of small RNA molecules that play an important role in the regulation of gene expression. Research into non-coding RNAs in particular miRNAs is currently one of the hottest yet challenging areas of molecular biology, in part due to the complex biogenesis of this class of molecules. Two main properties of these molecules, namely their length: being only ~22 nucleotides long and the high frequency of homology in each miRNA family contribute to making their analysis technically challenging.

This analysis requires highly specific assays that will allow reliable detection of individual members and also discriminate between mature (active) form and its precursor. In this presentation I will provide an overview of approaches currently utilised in miRNA analysis and will focus on two of them:

1) the locked-nucleic-acids based oligonucleotide microarray platform “miChip”, which has been developed in partnership with Matthias Hentze’s group and Exiqon, and qPCR for validation of microarray results and additional profiling of miRNAs detected by the miChip.

I will also discuss several points concerning preparation of samples for miRNA profiling in order to obtain robust profiles of miRNA expression.

Validation of Hits from an siRNA Library Screen Using Real-Time qPCR.

Shipley G.L.

The University of Texas Health Science Center-Houston, USA

Email: gregory.l.shipley@uth.tmc.edu

The use of siRNAs as a method to interrogate one or more key members of specific pathways involved in a biological process has become an invaluable tool. Working with a handful of siRNAs can easily be accomplished using classical laboratory procedures. However, with the advent of siRNA libraries targeting key classes of transcripts (kinases, phosphotases, nuclear receptors, etc.) not to mention genome wide siRNA libraries, the questions being asked and the methods required to perform large siRNA screens requires a different experimental approach. Ideally, one wants to be able to measure the biological process using either a biochemical or image-based assay while simultaneously measuring the degree of transcript knockdown by real-time qPCR. The most efficient way to screen the large numbers of siRNAs found in these libraries requires automation. However, the introduction of automation can present new challenges. Using an AR-YFP stable HeLa cell line, we have made great strides in automating the siRNA screening process for both an optimal biological assay and real-time qPCR readout. The challenges presented by this project and the solutions we have developed will be presented.

(micro)RNAome of human germ cell tumors: pathological and clinical implications.

Looijenga L.

Erasmus Medical Center Rotterdam, Netherlands, The

Email: l.looijenga@erasmusmc.nl

MicroRNAs (miRNAs) belong to the group of so-called non-coding RNAs, which function as endogeneous triggers of RNA interference (RNAi). RNAi is a natural cellular mechanism by which a hybrid between a mRNA and multiple miRNAs, resulting in double-stranded RNA sequences, are recognized as “foreign”. This results in degradation and/or inhibition of translation, thereby modulating the formation of protein. It has been demonstrated that proper embryonic development depends on a well-organized temporal and spatial expression of miRNAs, and many miRNAs are expressed in a tissue- and developmental stage-specific manner. miRNA also play a role in cancer development, and can act as oncogenes or tumor suppressor genes. Inactivation of P53 is supposed to be one of the crucial steps in the process of malignant transformation, because it allows cells to overcome cellular senescence, an irreversible stage of cell cycle arrest. This is supported by the observation that P53 is frequently inactivated in solid cancers. There are a limited number of exceptions to this rule, including the type II GCTs, i.e., the seminomas and nonseminomas. These originate from carcinoma in situ (CIS)/intratubular germ cell neoplasia unclassified (ITGCNU), being the counterpart of a primodial germ cell/gonocyte. The invasive tumors mimic early embryogenic development, and are in fact totipotent in nature. Overall, the type II GCTs contains wild type (WT) P53, which has been an issue of debate for years. This seemingly discrepancy between the need of P53 inactivation to allow the process of malignant transformation, and the P53 status in type II GCTs, is recently elucidated by us based on expression of a specific miRNA cluster (hsa-miR 371-373). These miRNAs are able to overrule the presence of wild type P53 in type II GCTs as well as derived cell lines. This allows the cells to overcome cellular senescence after oncogenic stress during their process of malignant transformation. This is another example of the impact of specific miRNAs in the process of malignant transformation. Based on this finding, we initiated a high-throughput expression analysis using quantitative PCR of a set of 157 as well as the 8 times 48 pools miRNAs in various types of GCTs and controls. The data obtained confirm the previous observation on hsa-miR 371-373, and demonstrate that the various histological GCT elements are, besides on the level of mRNA, also characterized by a specific pattern of expression of miRNAs.

Whole miRNA Profiling from Single Embryonic Stem Cell and early embryos.

Lao K.1, Tang F.2, Xu N.1, Livak K.1, Straus N.1, Surani A. M.2

(1) Applied Biosystems, California, US; (2) University of Cambridge, Wellcome CRC Institute, UK

Email: laokq@appliedbiosystems.com

MicroRNAs are short (17-25 nucleotides), non-coding RNAs that play critical roles in gene regulation and cellular differentiation during development. Recently developed miRNA micro-array techniques have contributed greatly to miRNA research but require too large an RNA sample to be used in many crucial studies, such as developmental studies involving primordial tissue samples from embryos or laser captured samples from developing tissues. Here we report a real-time PCR-based, 330-plex miRNA, expression profiling method that is sensitive and accurate enough to profile miRNA from samples as small as a single cell, such as an Embryonic stem cell. This technique will greatly facilitate miRNA-related research on stem cells and early embryos. It should also be of use in studies on carcinogenesis where only limited amounts of material is available from tissue biopsies or archived material.

Multi-Discipline Analysis of Gene Silencing: Complimentary use of Multiplex RT-qPCR, 2-D electrophoresis and Western blotting for RNAi based pathway analysis.

Teresa Rubio1, Katrina Academia2, Ning Liu2, Tim Wehr2, Steve Freeby2, Joseph Terefe1, Todd Yeck1, Aran Paulus2, Eli Hefner1 and Keith Hamby1, 1Bio-Rad Laboratories, Gene Expression Division, Hercules, CA and 2Bio-Rad Laboratories, Germany

Email: eli_hefner@bio-rad.com

RNA interference (RNAi) is a powerful tool used to modulate gene expression and to determine gene function. The activation of an RNAi pathway via delivery of small interfering RNAs (siRNA) into cells can result in the sequence-specific degradation of a messenger RNA (mRNA) and reduction of its corresponding protein product. The design of effective siRNA sequences in conjunction with efficient cellular delivery makes this a preferred tool for studying silencing and its effects. Analysis of gene specific silencing and subsequent changes in the expression of related genes and proteins is performed by assessing the levels of corresponding mRNAs or protein products. In this study, we demonstrate the effective downregulation of the cytoskeleton protein, β-actin, using specific 27-mer siRNAs and siLentFect Lipid Reagent in Hela cells. Subsequently, making use of 2-dimensional gel electrophoresis (2-DGE), Capillary LC-Nanospray and MS-MS, Western blotting and RT-qPCR we examine changes of expression profiles in response to β-actin gene silencing. We will present results of our analysis, showing changes in the expression level of several proteins, either directly or indirectly associated with actin filament function. Specifically, cofilin, an actin filament-disassembling factor, is found to be highly phosphorilated after b-actin downregulation. The complimentary use of these techniques facilitates rapid validation of 27-mer siRNA delivery and efficacy, screening for global changes in protein expression and multiplex validation of targets using qPCR techniques.

Comparison of endogenous control genes for normalisation of relative quantitative real-time PCR data in a study characterising microRNA expression in human breast cancer tissues.

Davoren P, Miller N, Lowery A, Mc Neill R, Kerin M.

National Breast Cancer Research Institute, Department of Surgery, Clinical Science Institute, University College Hospital, Galway, Ireland

Email: pamela.davoren@gmail.com

MicroRNAs act to negatively regulate gene expression at the transcriptome level. Aberrant expression of microRNAs has been observed in human proliferative diseases such as breast carcinomas, suggesting a possible role for these molecules in the regulation of tumour suppressor genes and oncogenes. Real-time quantitative PCR (q-PCR) is now being applied to the profiling of microRNA gene expression. To correct for variables such as amount of starting template and enzymatic efficiencies, q-PCR data is commonly standardised to an endogenous control gene which has stable expression across the sample set. Validating an endogenous control gene in the context of the relevant experimental settings is necessary to ascertain whether the level of expression of that gene varies beyond an acceptable level between samples.

This study aimed to validate the use of an endogenous control gene for the normalisation of microRNA q-PCR data in human breast cancer tissues. The expression of five microRNAs (let-7a, miR-10b, miR-16, miR-21 and miR-26b) and two small nuclear/nucleolar RNAs (RNU48 and Z30) was examined across 26 breast tumour samples, 5 benign breast tissues and 5 normal breast tissues. A 2-step q-PCR reaction was used, the reverse transcription step utilising a stem-loop primer specific to each candidate RNA and the PCR step employing TaqMan probes (Applied Biosystems). All q-PCR was performed on the ABI Prism 7000 Sequence Detection System. Intra- and inter- assay variations were all ≤ 0.3 standard deviations of a cycle threshold value. The two most stably-expressed candidate endogenous control genes, as determined by GeNorm (1) and Normfinder (2), were used to normalise the qPCR data for the target gene miR-30a-3p.

GeNorm and NormFinder both identified the same single endogenous control gene as being the most stably-expressed. The analysis methods differed in their secondary choice of genes, perhaps indicative of the different models used in each system. The relative quantity of miR-30a-3p did not differ significantly when normalized using the most stable gene alone or using NormFinder’s best combination of genes (p>0.05). Using the most stable gene alone and using GeNorm’s recommended combination of genes for normalisation did significantly change the relative quantity of miR-30a-3p (p=0.001) so consideration should be given to using this combination of genes for a more robust normalisation.

Characterization of miRNA expression in hESC lines using NCodeTM SYBR GreenER miRNA qRT-PCR.

Uma Lakshmipathy1, Mark Landers1, Sam An1, Brandon Nelson2, Mark Mercola2, Ron Hart 3, and Christopher Adams1

1Invitrogen Corporation, Carlsbad, CA, 2Burnham Institute for Medical Research, La Jolla, CA and 3Rutgers University, Piscataway, NJ

Email: mark.landers@invitrogen.com

MicroRNAs (miRNAs) are 19-25 nt non-coding RNAs that regulate gene expression by inhibiting translation or triggering degradation of specific mRNA targets. miRNAs appear to play a critical role in directing cellular differentiation. Unique microRNA expression profiles can be associated with specific cell types and stages of cellular development. Further, miRNA expression profiles can vary between undifferentiated cell lines. The NCode™ miRNA Analysis Platform provides an integrated solution for miRNA profiling including tools for miRNA purification, amplification, qRTPCR quantitation, a multispecies microarray and labeling kit. Using the NCodeTM SYBR GreenER miRNA qRTPCR system , we validated global miRNA expression profiles from several hESC lines in comparison to each other, hEC lines and their embryoid bodies. We were able to quantitate changes in miRNA expression from various cell lines by determining the fold difference from hEC values for critical differentiation markers. Additionally, we profiled differentiated cardiac cell lines vs. their undifferentiated progenitor cells by qRTPCR to validate cardiac specific miRNA expression patterns.


High-throughput RNAi Phenotype Analysis for Cancer Drug Target Identification and Validation by qPCR.

Sukru Tuzmen, Cumhur Ekmekci, Pinar Tuzmen, Felisa Blackmer, Holly Yin, Quick Que, Jeff Kiefer, David Azorsa, and Spyro Mousses

Translational Genomics Research Institute (TGen), United States of America

Email: stuzmen@tgen.org

Quantitative real-time PCR (qPCR) is now widely used owing to its simplicity, wide dynamic range of quantification, sensitivity, and precision, for accurate evaluation of gene expression. We describe here a qPCR process to validate specific gene silencing triggered by short interfering RNAs (siRNAs). RNA interference (RNAi) has become a widely used tool for determining the correlation between loss-of-function phenotypes and individual genes. Commercially available RNAi libraries have made high-throughput genome-scale screening a feasible methodology for studying mammalian cell systems. However, it is crucial that any observed phenotypic change be confirmed at either the mRNA and/or protein level to determine the validity of the targeted genes. Mimicking the natural gene silencing mechanisms of RNA interference (RNAi), synthetic siRNAs can be used to induce sequence-specific degradation of transcripts homologous to siRNAs. We have used synthetic siRNAs (Qiagen Inc., Germantown, MD) to study the function of genes by transfection into mammalian cells in a high-throughput manner. For siRNAs that trigger a phenotype, we used qPCR assays that have been designed specifically for validating transcript levels following RNAi based gene silencing. The TaqMan fluorogenic 5’ nuclease assay (ABI Inc.) has been found to be convenient, and sensitive for our target validation. Our results indicate a batch-to-batch consistency of this assay as well as consistency among sample replicates. In conclusion, this study presents an efficient and a reproducible method to validate RNAi gene knockdowns.

A new method for separation and characterization of Small RNA by On-Chip Electrophoresis.

Martin Greiner 1, Marcus Gassmann 1, Marc Valer 2, Hans Brunnert1

1Agilent Technologies, Waldbronn, Germany; 2Agilent Technologies Inc., Santa Clara, USA

Email: martin_greiner@agilent.com

A multitude of microRNAs has 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 and understanding on how RNA integrity and different purification protocols affect its qualitative and quantitative analysis.

Here we describe a novel Microfluidic assay that is able to perform very sensitive high resolution analyses of small RNA samples on a commercial lab-on-a-chip platform commonly used for RNA QC analysis. The assay delivers information about size and concentration of small RNA species like miRNA, siRNA, t-RNA etc, in the range from 10 to 150nt. Purified or enriched small RNA fractions, as well as total RNA samples with miRNA concentrations down to 50 pg/µl can be analyzed.


Main Session:   Single Cell qPCR

Chair:   M. Kubista / B. Rocha

Lecture hall   HS 14

Large Scale Cell-to-cell Variations in Gene Expression.

Raj A and Tyagi S.

Public Health Research Institute, Newark NJ, United States of America

Email: sanjay@phri.org

Biologists usually grind up a tissue, extract its RNA and obtain its gene expression profile. These profiles represent the average number of mRNA molecules present in each cell. We have found that the numbers of mRNA molecules expressed by different cells of an identical genotype are so different from each other that very few cells correspond to the reported averages. Using an in situ hybridization procedure that has a single molecule resolution we were able to explicitly count the number of molecules of specific mRNAs produced in each cell in a population of cells. We found evidence for large-scale cell-to-cell variations. Akin to the gene expression "noise" previously reported in the prokaryotes and yeast, these variations stem from the nature of transcription in higher eukaryotes, which our results indicate, occurs in bursts. Randomness in the onset and dissipation of these bursts of mRNA synthesis, in combination with the short life-time of mRNA, results in these variations. The bursts of mRNA synthesis in different genes occur independently of each other. The origins of this stochastic mRNA synthesis may lie with the unique mechanisms that open up chromatin context of the gene and render it conducive for mRNA synthesis and later sequester the gene to turn off the synthesis.

If the magnitude of the observed variations is so large then how are cells are able to maintain their relatively constant phenotypes? Part of the answer is that proteins generally stay around in the cell longer then the mRNAs do. The preexisting pools of proteins receive periodic inputs from the transient bursts of mRNA production. Since the size of the protein pools is relatively large it is buffered against the variations in mRNA levels. Thus the levels of proteins vary less then the levels of mRNAs between cells. However, the life-times of proteins vary a lot and the levels of short-lived proteins must be determined by the variations in the levels of their respective mRNAs. To cope with such variations organisms may have developed other yet unidentified mechanisms. In some situations these variations will even be beneficial, as they will serve as an extragenetic substrate for adaptation to the transient variations in the environment.

Single cell microRNA and mRNA profiling reveals global gene expression changes during mouse ES differentiation.

Ruoying Tan 1, Leila Bahreinifar 1, Dana Ridzon 1, Karl Guegler 1, William Strauss 2, Caifu Chen 1

1Applied Biosystems, 850 Lincoln Centre Dr., Foster City, CA 94404, USA and 2Department of Molecular, Cellular, & Developmental Biology, University of Colorado, Boulder, CO 80309, USA

Email: Ruoying.Tan@appliedbiosystems.com

We describe a new method for simultaneously quantifying 237 mouse microRNA (miRNA) and 21 messenger RNA (mRNA) genes from each of 70 single cells. The method is based on multiplex RT, multiplex preamplification, and singleplex real-time TaqMan® PCR assays. Assays are quantitative for > 3-log dynamic range. Single cell expression signature could classify individual ES, embryoid body (EB), and somatic cells. Significant inter-cell variations of both miRNA and mRNA expression were observed within or between ES cell lines, indicating the heterogeneity of ES cells. Highest variability was observed among EB cells (CV = 139%), demonstrating that EB cells undergo differentiation at different stages. Interestingly, expression of ES marker gene OCT4 and signaling gene Tdgf1 was absent in 3T3 and splenocyte cells, highly expressed in ES cells, and significantly reduced in EB cells. Furthermore, there is no correlation in expression levels between miRNAs and their predicted target mRNAs, supporting translational repression model. Our results gain new insight of both miRNA and mRNA expression patterns at a single cell level.

Duplex RT-LATE-PCR reveals transcript gradients in sets of single cells recovered from 8-cell mouse embryos.

Cristina Hartshorn, Odelya Hartung and Lawrence J. Wangh.

Biology Dept., Brandeis University, Waltham, MA, USA

Email: hartcris@brandeis.edu

The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass (ICM) are pluripotent and express transcripts such as Oct4 RNA, which are down-regulated in the surrounding, differentiated trophectoderm (TE). Conversely, other genes are active in the TE and silenced in the ICM. These include Xist (expressed only in females) and Cdx2 (in both sexes). Prior to blastocyst formation, all these RNAs are ubiquitously found in blastomeres of embryos at the 8-cell stage. It is plausible, however, that transcript levels differ among blastomeres of the same embryo, and that these quantitative differences may presage the fate of their daughter cells. Testing this hypothesis presents numerous technical challenges because it requires simultaneous quantification of different RNAs in sets of single cells isolated from the same embryo. We have overcome these difficulties by combining PurAmp, a single-tube method for RNA preparation and quantification, with LATE-PCR, an advanced form of asymmetric PCR. We initially constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy using both biological samples and analysis of the LATE-PCR fluorescent signals. The linear slope of these signals is a sensitive tool to establish that amplification has been achieved with comparable efficiency for all templates analyzed. The Oct4/Xist duplex was an ideal test system, because comparison of data from males and females allowed us to determine that, due to the properties of LATE-PCR, Oct4 amplification was unaffected by sex-related differences in Xist expression (females: Oct4 +, Xist ++; males: Oct4 +, Xist -).

Our results show that both Oct4 and Xist RNA levels vary in individual blastomeres comprising the same embryo, with some cells having particularly elevated levels of either transcript. This is significant because all cells in the 8-cell embryo are believed to be developmentally equivalent. Our data also indicate that Xist and Oct4 expression levels are not correlated at this stage, although transcription of both genes is up-regulated at this time in development. We have now developed an additional assay for simultaneous measurements of Oct4 and Cdx2 RNA, in light of recent findings that these two genes are reciprocally regulated.

This work describes the first example of RT-LATE-PCR and its utility for single-tube, multiplex quantitative analysis of transcripts in single cells. Levels of different RNAs can be accurately measured independently of their relative abundance; this is not possible with symmetric PCR. The techniques illustrated here are widely applicable, for instance to gene expression analysis in stem cells and cancer cells and to preimplantation genetic diagnostics. We are also employing these strategies for multiplex quantitative end-point detection of RNA viruses.

Gene expression or SNP profiling from picograms of RNA using Multiplexed Tandem PCR.

Stanley K.

Corbett Life Science, Australia

Email: keith.stanley@corbettresearch.com

MT-PCR is a 2-step PCR process for gene profiling or mutation detection. It can be configured to produce a gene expression profile of up to 96 genes from 50 ng of RNA down to 10 pg of RNA enabling gene profiles to be obtained from a single section of formaldehyde fixed paraffin embedded specimens (1 to 10 ng) or from single cell amounts of RNA (about 10 pg). Separating the PCR reaction into 2 steps allows optimum conditions to be used for amplification of rare templates during the multiplexed preamplification cycles and conditions that minimise primer dimer formation during the quantification stage.

Expression is first normalised against one or more housekeeping genes included on the MT-PCR-disc, and then between genes on the experimental and control discs. The correlation of MT-PCR gene expression measurements to qPCR results, or of MT-PCR results with 100 fold different amounts of input RNA were both > 0.9. The coefficient of variation between assays performed on different days was 3% (in the Ct value) for cell line RNA (10 experiments) and 8% for FFPE sections (5 different sections).

When coupled with High Resolution Melt analysis, MT-PCR can be used to perform multiplexed SNP analysis from single cell quantities of RNA.

Quantitative RT-qPCR of individual dopaminergic neurons from vital and fixed tissues.

Birgit Liss

Physiology, Philipps University of Marburg, Germany

Email: liss@staff.uni-marburg.de

Dopaminergic (DA) midbrain neurons are arranged within two overlapping nuclei, the ventral tegmental area (VTA) and the more lateral substantia nigra (SN). Their function is crucial for the control of voluntary movement, reward-based behavioral decision making as well as for cognition and memory. Consequently, selective degeneration or functional dysregulation of DA neurons are causally involved in common human disorders like Parkinson disease (PD), drug addiction, and schizophrenia. We aim to identify differentially expressed genes and related cellular mechanisms that define different physiological and pathophysiological functions in specific subpopulations of DA midbrain neurons.

We currently focus on molecular mechanisms that determine the differential vulnerability of DA neurons, a hallmark of Parkinson disease and its chronic animal models. To analyse dopaminergic function and gene-expression at the level of the individual neurons, we combine electrophysiological patch-clamp techniques in living mouse brain slices, or UV-laser-microdissection (LMD) from fixed human or mouse brain-cryosections with quantitative real-time RT-PCR approaches. In a hypothesis-driven approach, we focus on the role of ion channels in the pathophysiology of the DA midbrain system. Already under physiological control conditions DA neurons in SN and VTA display a variety of distinct electrophysiological properties, which are correlated with qualitative and quantitative differences in mRNA expression-levels of related ion-channel subunits. Furthermore, we could demonstrate that differences in ion channel expression and activity are crucial for the differential survival of DA midbrain neurons in chronic neurodegenerative disease. In particular, we showed that the selective electrical silencing of DA SN neurons via ATP-sensitive potassium (K-ATP) channels was correlated with higher mRNA expression-levels of K-ATP channel subunits and lower mRNA levels of the mitochondrial uncoupling protein UCP-2. To identify candidate genes for differential vulnerability of DA neurons in an unbiased manner, we carried out single-cell microarray studies that resulted in a list of about sixty candidate genes that were differentially expressed between individual SN and VTA DA neurons. We successfully validated the majority (75%, n=15 of 20) of selected candidate genes from our single-cell microarray studies by defining their expression levels in selective and non-amplified cDNA pools of laser-microdissected SN and VTA DA neurons, respectively via quantitative RT-PCR. In a complementary approach, we are currently studying quantitative gene expression of human SN DA neurons from post mortem brains of idiopathic PD-patients and respective matched controls by combining LMD and real-time PCR as described above. Data of these studies will be presented and methodological issues for RT-qPCR analysis in particular of human post mortem material will be discussed.

Intracellular expression profiles in the Xenopus laevis oocytes revealed by quantitative real-time PCR.

Radek Sindelka 1, Jiri Jonak 1, Rebecca Hands 2, Stephen A Bustin 2, Mikael Kubista 1,3

1IMG AS CR, Czech Repbulic, 2Institute of Cell and Molecular Science, Royal London Hospital, United Kingdom and 3TATAA Biocenter, Sweden

Email: sindelka@img.cas.cz

Cell determination during early development directly depends on mRNA and protein cell content and distribution. In mammalian cells, mRNA profiling is limited by small amounts of RNA. In contract, a Xenopus egg contains very large amount of mRNA, which opens for expression studies on the sub cell level. Here, we quantified mRNA levels of several selected maternal genes in different sections of the Xenopus egg. We determined the amount of these mRNAs in egg sections along the animal-vegetal axis by real-time RT-PCR. The experiments were performed on eggs before and after fertilization. Based on these results a 3D map of key mRNAs in a single Xenopus egg cell will be constructed, that will give clues to the relation between mRNA distribution and cell division and ultimately differentiation.

Systematic Analysis of single cells by PCR.

Mann W.

Advalytix AG, Germany

Email: mann@advalytix.de

Advalytix has developed a new single cell amplification platform. The AmpliGrid is a microscope slide with a chemically modified surface in order to define 48 reaction centers based on hydrophilic / hydrophobic structuring. On each of the reaction sites a 1µl amplification reaction can be set up. In contrast to conventional tube assays single cells can be deposited and quality checked immediately before the amplification reaction (that might be PCR, RT-PCR, etc). Combining HT methods like FACS sorting result in a systematic genetic analysis of single cells for various applications. Based on the systematic analysis of single cells Advalytix has developed a technique called ABC (amplification based counting) that makes use of the fact that in multiplex PCR there is a correlation between the number of drop outs and the number of identical target sequences that have been introduced as starting material. In single cells it is now possible to distinguish discrete numbers of DNA or RNA sequences in an absolute manner.

Detection and quantification of mRNAs in single human embryonic stem cells.

Ståhlberg A. Bengtsson M. Semb H.

Stem Cell Center, Lund University, Sweden

Email: anders.stalberg@med.lu.se

Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of the blastocyst. They are unique self-renewing cells that have the capacity to generate any cell type in the body. This capability provides the basis for considering the hESCs as an unlimited source of cells for replacement therapies and for the treatment of a wide range of diseases such as diabetes mellitus, Parkinson and Alzheimer diseases. Our aim is to develop new research tools to enable a better validation and understanding of the sequential differentiation of pluripotent hESC into definitive endoderm, pancreatic β-cell precursors, and finally -cells. Undifferentiated hESCs requirebinto terminally differentiated  expression of NANOG, POU5F1 (also known as OCT4) and SOX2 to maintain their unique characteristics. To date most transcriptome analyses on hESCs have relied on measurements from cell populations, thereby not revealing how the expression of NANOG, POU5F1 and SOX2 influence differentiation at a level of single cell. To further our understanding of the means by which these transcription factors control the pluripotency and self-renewal of hESCs, we have performed quantitative gene expression studies of individual cells. Accurate single cell gene expression measurements require sensitive and robust assays. We have evaluated and optimized all steps from cell collection to data analysis for accurate single cell gene expression measurements. We apply this method for investigation of population heterogeneity, single cell correlations and transcript distributions.

Molecular portraiting of normal and tumor human breast stem cells.

Pece S.1, Confalonieri S.2, Vecchi M.2, Matera G.1, Ronzoni S.1, Tizzoni L.2, Bernard L.2, Pelicci P.G.1, and Di Fiore P.P.2

1IEO (Istituto Europeo di Oncologia), Milan, Italy and 2FIRC Institute for Molecular Oncology (IFOM), Milan, Italy

Email: salvatore.pece@ifom-ieo-campus.it

A prediction of the stem cell theory of breast cancer is that complete elucidation of the normal stem cell biology will be instrumental to gain insights into breast carcinogenesis. Given the lack of specific human breast stem cell markers, we devised a strategy based on the ability of breast stem cells to generate clonally-derived ‘mammospheres’ ex vivo, in combination with the use of a ‘surrogate’ marker, the PKH26 cell linker, which stably incorporates a fluorescent dye into the lipid regions of the plasma membrane. The strength of this approach is that stem cells are not defined phenotypically, i.e. using cell surface markers, but functionally through their intrinsic property to be slow-dividing. In fact, they accumulate the PKH26 dye and remain the most intensively fluorescent cells within mammospheres, whereas their actively dividing and differentiating progeny progressively loose fluorescence through dilution of the membrane-bound dye. We used FACS analysis to sort the most highly fluorescent cells, comprising the strict-sense stem cells, from the progeny of committed progenitors. Functional analysis of the different fractions confirmed that only the PKH26+ cells exhibited key defining features of ‘stemness’, such as: i) self-renewal property (re-formation of mammospheres upon serial passages in vitro); ii) ability to generate both epithelial and myoepithelial histotypes, as determined with specific lineage markers; iii) formation of alveolar/ductal-like outgrowths in vitro. Using oligonucleotide-based arrays (Affymetrix), we obtained trascriptional profiles of the two populations separated by their differential epifluorescence. The comparative analysis of genes differentially regulated by a factor ≥2 uncovered many distinguishing features between the two PKH26 populations, strengthening the notion that we indeed separated stem cells from their differentiating progeny. Selected candidate hits were further validated by Q-RT-PCR using a multiplex reaction with pooled 96 Taqman gene expression assays (Applied Byosystem) as a source of primers in a pre-amplification reaction, followed by a Taqman low density array experiment. This approach confirmed that genes associated with an immature and quiescent state (‘stemness’) were associated with PKH26+ cells. In contrast, PKH26- cells over-expressed transcripts associated with proliferation, cell cycle progression and checkpoint control together with markers of myoepithelial/epithelial differentiation. In conclusion, we set up a strategy for the functional characterization and molecular portraiting of normal (and tumor) breast stem cells, with the ultimate goal to highlight the molecular mechanisms controlling normal and aberrant morphogenetic programs.

Quantification of multiple gene expression in individual cells.

Antonio Peixoto, Marta Monteiro, Benedita Rocha, Henrique Veiga-Fernandes

INSERM U591, Faculty of Medicine Paris 5 René Descartes, France

Email: rocha@necker.fr

Quantitative gene expression analysis aims to define the gene expression patterns determining cell behavior. So far, these assessments can only be performed at the population level. Therefore, they determine the average gene expression within a population, overlooking possible cell-to-cell heterogeneity that could lead to different cell behaviors/cell fates. Understanding individual cell behavior requires multiple gene expression analyses of single cells, and may be fundamental for the understanding of all types of biological events and/or differentiation processes. We here describe a new reverse transcription-polymerase chain reaction (RT-PCR) approach allowing the simultaneous quantification of the expression of 20 genes in the same single cell. This method has broad application, in different species and any type of gene combination. RT efficiency is evaluated. Uniform and maximized amplification conditions for all genes are provided. Abundance relationships are maintained, allowing the precise quantification of the absolute number of mRNA molecules per cell, ranging from 2 to 1.28 x 10(9) for each individual gene. We evaluated the impact of this approach on functional genetic read-outs by studying an apparently homogeneous population (monoclonal T cells recovered 4 d after antigen stimulation), using either this method or conventional real-time RT-PCR. Single-cell studies revealed considerable cell-to-cell variation: All T cells did not express all individual genes. Gene coexpression patterns were very heterogeneous. mRNA copy numbers varied between different transcripts and in different cells. As a consequence, this single-cell assay introduces new and fundamental information regarding functional genomic read-outs. By comparison, we also show that conventional quantitative assays determining population averages supply insufficient information, and may even be highly misleading.

Quantitative PCR of heterogeneous tissue: Lessons from the islets of Langerhans.

Martin Bengtsson1, Anders Ståhlberg1, Patrik Rorsman2

1Lund University, Sweden and 2University of Oxford, UK

Email: martin.bengtsson@med.lu.se

The islets of Langerhans reside in the pancreas where they serve as the glucose sensor of the body, making sure the blood glucose concentration stays within a healthy range. This regulation fails in patients with diabetes. Each islet consists of approximately 1000 cells, of which ~80% are insulinproducting b-cells, ~20% glucagonsecreting a-cells and ~5% somatostatinreleasing d-cells. The functions of these cells are fundamentally different, and studies of them require either cell sorting or single cell analysis. In our search to reveal cellular mechanisms and find possible therapeutic targets for diabetes treatment we developed a method for quantitative mRNA measurements in individual mammalian cells using RT-PCR. We took great care to maximize the robustness, reliability and efficiency of the method. For example, we evaluated the need for – and choice of – cell lysis buffer and heat treatment. We investigated the accuracy of the quantification, thereby defining a limit at which the technical variation exceeds the biological variation. These results, together with data from glucose stimulation of islet cells, will be showed in the presentation.

Islet cells are electrically active. Ion channels play a fundamental role in hormone release and several types of diabetes are caused by dysfunctional ion channels. In collaboration with Applied Biosystems, we combined electrophysiological recordings, single cell collection and the TaqMan® PreAmp Master Mix Kit on individual islet cells. The preamplification allows us to measure hundreds of genes from a single cell, and we investigated the isoforms of sodium ion channels in a- and b-cells. The results revealed cell-type specific expression of sodium channel isoforms and correlations to Na2+-currents measured with patch-clamp.

Quantitative real time PCR for single tumor cell based diagnostics.

Kemming, D. Meyer-Staeckling, S. Alpers, I. Brandt, B.

UKE Hamburg, Germany

Email: d.kemming@uke.uni-hamburg.de

One third of annually 50,000 newly diagnosed breast cancer patients will die from metastases. However, the individual risk for metastases is still estimated statistically by parameters determined on the primary tumor yet. We and other research groups have shown that breast cancer patients harbor single disseminated tumor cells (DTC) in their bone marrow or peripheral blood even in the absence of lymph-node involve-ment (stage N0) or distant metastases (stage M0) at the time of primary surgery. The detection of aberrations of key oncogenic genes could enhance the specificity and may lead to the identification of the potential founder cells of metastases.

In consequence, we developed a method for the isolation and real time PCR based characterization of tumor cells. The cells to be analyzed are transferred under micro-scopic control selectively on hydrophobic coated glass slides carrying lysis buffer in a spot. The surface ensures that the genetic material can be completely removed after the cell lysis. This lysate is subsequently subjected to isothermal whole genome am-plification (WGA). As a test system MDA-MB-468 and SK-BR-3 cells were used, ge-nomic leukocyte DNA was used as reference material. MDA cells show a strong am-plification of the EGF receptor gene while SK-BR-3 cells show a low level amplifica-tion of this gene. Single cell WGA products were used to analyze EGFR gene ampli-fication by real time PCR. The copy number of the SOD2 gene was used as refer-ence. In order to test if WGA products reflect the genomic situation of the tumor cells, EGFR gene copy numbers were compared between WGA products and tumor cell DNA. Using two different cell lines, we were able to determine similar genomic altera-tions in the WGA products and in the unamplified material.

The specificity of the method was confirmed by sequencing and microsatellite analy-ses. A cell of a cell line harbouring two known mutations in the tp53 gene was sub-jected to the single cell picking/WGA procedure. Both mutations were found using the genetic material isolated from the single cell. PCRs targeting microsatellites on chro-mosome 7, 8, 10, 13, 16 and 17 were performed to ensure the amplification of the entire genome.

Therefore this method might be suitable for more detailed examinations of bone marrow and blood from cancer patients.

Heterogeneity in complex tissues identified by quantification of nucleic acids in single cells.

Philip Day1,2, Lin Chen1, Pierre-Alain Auroux2, Stephan Mohr2, Nicholas Goddard2, Andreas Manz1 and Peter Fielden2.

1Analytical Sciences, ISAS, Dortmund, Germany and 2University of Manchester, UK

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

Real-time quantitative and conventional end-point polymerase chain reaction (PCR) are ubiquitously applied in gene-based assays, however routine reproducibility is limited by intrinsic technical limitations. These restrictions include poor compatibility to study low transcript number amongst a high background of other nucleic acids, analysis of several targets from minute biopsy samples, single cell analyses, plus additional constraints relating to time, expense and risk of cross-contamination.

PCR micro total analysis systems (μTAS) devices can circumvent these drawbacks. Two uTAS concepts are to be presented. In the first, a novel approach is based on a shunting system where an aqueous sample plug is shunted from one temperature zone to another by a syringe pump system, and benefits from reaction parallelisation. A second more advanced system engages the application of flowing streams of aqueous nanolitre droplets for use in single cell PCR. Cell lysis, reagent mixing, thermal cycling and microfabricated real-time optics have been developed. These will ultimately derive μTAS devices for very high throughput uses and more robust inter-laboratory standardisation.

Data is presented revealing that quantification of nucleic acids via PCR has a mandatory requirement to employ miniaturisation, and that this will assist in functionally relating numbers of copies of analyte nucleic acid to single cell type and decipher the cell composition of heterogeneous tissues.

Session:   Immuno - qPCR

Chair:   HHD. Meyer / C. Niemeyer

Lecture hall   HS 14

Immuno-qPCR: Novel Opportunities in Clinical Diagnostics and Research.

Niemeyer C.

Universität Dortmund, Germany, FB Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Str. 6, D-44227 Dortmund, e-mail: christof.niemeyer@uni-dortmund.de

Email: sekretariat-bcmt.chemie@uni-dortmund.de

The Immuno-qPCR technology combines the advantages of flexible and robust immunoassays with an exponential signal amplification, typical for PCR [1]. Immuno-qPCR is based on chimeric conjugates of specific antibodies and nucleic acid molecules, the latter of which are used as markers to be amplified by PCR for signal generation. The enormous efficiency of nucleic acid amplification typically leads to a 100 – 10,000 fold increase in sensitivity, as compared with the analogous enzyme-amplified immunoassay. We have developed a proprietary detection and screening technology focused on the analysis of low abundant and hardly detectable biomarkers. This technology is being commercialized under the trademark Imperacer™ by the Dortmund based company Chimera Biotec. Imperacer™ services and products are designed for R&D, screening, monitoring, QC and a broad range of indications, such as CNS disorders, cancer, infectious diseases, auto inflammation, doping control, and others.

The lecture provides an overview on the scope and performance of the Imperacer™ Immuno-qPCR technology, including detailed discussions of experimental parameters, such as sensitivity and dynamic range, sample requirements, and tolerances against matrix effects in the detection of various biomarkers, including drug candidates, small-molecule hormones, cytokines, tumormarkers, viral proteins, and others.

Use of Immuno-qPCR for quantifying proteins in large-scale TAP-tag collections.

Lind K. and Norbeck J.

Chalmers University of Technology, Gothenburg, Sweden

Email: kristina.lind@chalmers.se

In recent years, the sequencing of several eukaryotic genomes has paved the way for truly large-scale experimental approaches. Both for our understanding of the biological process in cells and for the development of new drugs. Saccharomyces cerevisiae, was the first completely sequenced eukaryot and is a widely used model organism. Several genome-wide collections have been constructed in S.cerevisiae, e.g. a set of gene-deletions covering all non-essential genes, GFP-tag (Green Fluorescent Protein-tag) collections and TAP-tag (Tandem Affinity Purification-tag) collections for essentially all 6000 genes. The GFP-collection has been used in combination with flow cytometry (FACS) to quantify the expression of all tagged proteins. Similarly, the TAP-tag collection has been used in combination with western blotting for protein quantification. Both these approaches have serious drawbacks, the GFP-approach suffers from a rather high detection limit and the TAP-tag approach is limited by the difficulties associated with protein extraction and western analysis (e.g. difficulties in background subtraction and lack of linearity because of saturation of signal). Thus, there is a need for development of more sensitive and reliable protein quantification methods suitable for large-scale analysis.

We have adapted the immuno-qPCR assay, previously used by us for quantifying prostate specific antigen (PSA), for quantification/detection of the TAP-domain. Briefly, we utilize an affinity purified chicken antibody raised against protein A (which is present in two copies in the TAP-tag) as both capture and DNA-conjugated detection antibody. The immuno-qPCR-detection of the TAP-tag is a sensitive method with a large quantification range that combines the molecular specificity of antibodies with the DNA amplification power of PCR. Using real-time PCR there is no need for gel electrophoresis which makes the assay time short. A drawback however, compared to western blot analysis, is that no information about protein modifications or breakdown products is gained using immuno-qPCR. We envisage that our assay, when combined with automated sample handling, can be used to sensitively quantify essentially all yeast proteins under a variety of conditions. Furthermore, our TAP-assay is not restricted to use in yeast, the TAP-tag has proven its worth in organisms ranging from E.coli to humans.

With this newly developed assay we have analysed the expression of a number of proteins in yeast grown on YPD or YPD plus 1 M NaCl and compared it to published data from 2D-PAGE and a large scale western approach. The correlation to the 2D-PAGE data was good while the correlation to western data (restricted to YPD-growth) was more problematic. We will discuss the reasons for this.

Immuno-Real Time-PCR as a sensitive diagnostic tool: case of prion proteins.

Ruelle Virginie and ElMoualij Benaissa

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. Prion diseases are caused by tertiary conformational change of the normal form of prion protein (PrPc) in host cells to the pathologic form (PrPsc) and its accumulation in central nervous system2.

The conformational change of the pathologic form confers to it a partial protease resistance property being very convenient to distinguish the two prion forms, normal and pathologic. Actually, different rapid detection kits are approved by the European Community for a systematic diagnosis of bovine spongiform encephalopathy and scrapie, by screening the brain stem at the post-mortem stage3. These detection kits are very efficient to detect prion protein at the post-mortem stage and permitted to warrant the safety of the bovine and sheep production. However, other ultra-sensitive methods need to be developed to allow an early detection of prion protein for living animals.

In this study, the immuno-quantitative-Polymerase chain reaction (iqPCR)4 was applied to detect ultra-low levels of prion protein. 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 format5-6. To illustrate the advantages of iqPCR, we have compared it with a conventional ELISA and western blotting technique in experiments aimed at detecting the resistant form of prion protein in bovine and human brain extract. Using iqPCR, a minute quantity of prion was detected with a detection threshold at least 10-fold lower than classical techniques. The iqPCR could therefore be potentially useful for the detection of prion protein at early stage.

Feasibility of simultaneous measurements of mRNA expression and corresponding protein level in microdissected tissue samples by real-time technology: PSA in normal and tumour tissues as a demonstrative model.

Pamela Pinzani 1, Kristina Lind 2, Francesca Malentacchi 1, Francesca Salvianti 1, Mikael Kubista 3, Mario Pazzagli 1, Claudio Orlando 1.

1Department of Clinical Physiopathology, Clinical Biochemistry Unit, University of Florence, Italy, 2Department of Chemistry and Bioscience, Chalmers University of Technology, Gothenburg, Sweden and 3TATAA Biocenter AB, Gothenburg, Sweden

Email: p.pinzani@dfc.unifi.it

Laser assisted microdissection (LAM) has been introduced extensively in cancer molecular biology studies with the aim of selecting pure cell populations from heterogeneous tissues. New technologies in the field of LAM can thus overcome the problem of cellular heterogeneity that represents a significant barrier to the molecular analysis of normal and pathological tissue (Simone et al 2000). Moreover, it allows molecular analysis of cell populations in their native tissue environment and it may be potentially applicable to biopsies obtained in preoperative diagnostic procedures. Several attempts have been made in order to determine whether mRNA levels could be used to accurately predict protein levels in tissue sample extracts (Hiser et al. 2006). Results obtained for different proteins gave either positive or negative results (Gygi et al. 1999; Nicoletti et al. 2001) with an additional and intrinsic limitation due to the relative lack of sensitivity of the actual methods used for protein measurements.

Recently, immuno qPCR has been developed as a new tool for the measurement of proteins levels with up to 1000-fold increase of detection limit compared to the commonly used immunoassay. It is based on the use of a real-time PCR detection of the immuno-complex generated in the immunoassays. It has been applied to the measurement of protein in serum and plasma samples, but until now no evidence has been reported on microdissected tissues.

In this study, prostate-specific antigen (PSA) mRNA expression data were generated from microdissected primary prostate cancers and corresponding normal tissues by real-time RT-PCR. To calculate the expression of PSA mRNA in each microdissected sample, we referred to an external reference curve generated with synthetic cDNA obtained by cloning the target in the expression vector pcDNA3.1/CT-EGFP-TOPO (Invitrogen). PSA protein determinations were performed at Chalmers University of Technology (Gothenburg, Sweden) on tissue samples microdissected from areas of serial tissue sections paired to those used in gene expression measurements following the immuno qPCR procedure already reported by Lind K et al (2005). Linear regression analysis between PSA mRNA copies and protein molecules in normal and tumour samples was statistically significant for both sets of data. The Pearson coefficient resulted R=0.782 (p=0.006) for normal microdissected tissues and R=0.667 (p=0.041) for tumour microdissected samples. Confirming data regarding PSA mRNA expression and protein production have been obtained by using the prostate cancer cell line LNCap.

In conclusion, we demonstrated for the first time the feasibility of the simultaneous application of real-time RT-PCR and immuno qPCR to purified homogeneous cell populations obtained by LAM. These methods could represent a powerful tool to enhance the diagnostic value of gene expression studies in human cancers at mRNA and protein levels.


Session:   Pre-analytical-Steps

Chair:   A. Stahlberg / J. Huggett

Lecture hall   HS 15

An optimised protocol for extracting RNA from single bovine oocyte and blastomeres.

Marc Boelhauve1, Fabiola F Paula-Lopes2, Tuna Güngör1, Eckhard Wolf1

1Institute of Molecular Animal Breeding and Biotechnology, LMU Munich, Germany and 2Laboratório de Biotécnicas da Reprodução, Departamento de Medicina Veterinária da Universidade Federal Rural de Pernambuco, Recife – PE, Brazil

Email: m.boelhauve@gen.vetmed.uni-muenchen.de

Quantitative PCR (qPCR) analysis of gene expression in single bovine oocytes or blastomeres from early preimplantation embryos needs to meet optimized requirements for the isolation of RNA, reverse transcription reaction and even qPCR. In this study the first hurdle to a sufficient detection of low abundant genes was analyzed. There are many protocols available for extracting RNA from different materials, but normally large amounts of tissues are required. For extracting RNA from bovine preimplantation embryos several methods are available, but the RNA recovery per embryo is too low for the detection of low abundant genes. Therefore a modified/optimised isolation method is essential for obtaining sufficient RNA recoveries of single oocytes/embryos or even single blastomeres. Several experiments were conducted to increase the RNA recovery. In the first experiment different isolation protocols, taken from the literature, were compared (isolation of total RNA and messenger RNA). In a second experiment the best isolation protocol was used to analyse the influence of a coprecipitant (e.g. glycogen) on the RNA recovery and the inhibition of the reverse transcription process. In the third experiment the collection/storage of oocytes was compared (e.g. RNAlater or liquid nitrogen). Due to the low amount of RNAs, standards for the RNA isolation and the reverse transcription process were evaluated. Finally, total RNA concentrations were measured by two different methods (Nanodrop and Agilent Bioanalyzer) of ten bovine embryonic developmental stages (immature oocyte up to hatched blastocyst). For each experiment studied, RNA was reverse transcripted with Omni Extreme Script and random hexamer primers. The RNA recovery was analysed by the detection of transcript levels of high (STAT3), middle (Histone 2A) and low abundant (Leptin Receptor, LEPR) genes in an ABI PRISM SDS 7000 apparatus.

Laser microdissection – Bridging the gap between sample preparation and molecular biological analysis.

Hagen-Mann Kerstin

Carl Zeiss MicroImaging, Germany

Email: k.hagen-mann@zeiss.de

Targeting single cells, defined cells of a united cell structure or cell culture requires tools for precise selection of such targets. By means of laser microdissection and contact free catapulting into reaction tubes or onto glass surfaces researchers can prepare very homogeneous samples for gene expression, fingerprinting or pure cell cultures. We can prove that manipulation with laser microdissection does not affect the behaviour of cells in a cell culture, it does not change their characteristics (e.g. markers) and they survive this treatment easily. The target for laser microdissection can be an area of a tissue section, single cells of such a slide or even nuclei and single chromosomes or parts of them as well as living cells. Because the excision and transfer into the reaction tube is contact free, this technique provides a perfect contamination free environment for preparation of samples for all contamination sensitive downstream analysis applications. Reproducible sample preparation of defined numbers of cells are the prerequisite for quantitative experiments and their results.

Bring in the marines! Removal of contaminating DNA by marine enzymes in RT-PCR.

Elde M., Lanes O. and Gjellesvik D.R.

Biotec Pharmacon, Norway

Email: morten.elde@biotec.no

Marine enzymes from cold-adapted organisms exhibit properties that make them useful as tools in molecular biology. A common feature for these enzymes is high activity at low temperatures and that they are easily heat inactivated at moderate temperatures. Here we present the use of two different marine enzymes in RT-PCR.

The presence of contaminating DNA in quantitative RT-PCR is often a problem and can give erroneous results. The origin of the contaminating DNA may be genomic from the RNA source, or may be previous PCR products (carry-over contamination).

A nuclease from the arctic shrimp ( Pandalus borealis ) has properties that makes it useful for removal of contaminating DNA in a RT-PCR reaction. It is double-strand specific and easily inactivated. Here we show how the shrimp nuclease, in a one-step RT-PCR, removes double-stranded DNA from the RT-PCR mix, leaving RNA and primers intact.

Uracil-DNA-Glycosylase (UNG) is an enzyme often used to remove carry-over contaminants in quantitative PCR. We have tested the use of a marine UNG from cod ( Gadus morhua ) for carry-over prevention in RT-PCR, and compared it with other UNGs already available. Contaminating DNA is efficiently removed by the cod UNG but does not affect the sensitivity (Ct) of the qRT-PCR assay.

Multiplex preamplification of limited samples and novel analytical controls.

Zimmermann Bernhard, Wang Jianghua, Wong David

UCLA, Dental Research Institute

Email: bgz@ucla.edu

We present a new method for the multiplex 1-step RT-PCR based preamplification of mRNA that allows the determination of extensive expression patterns even from limited clinical samples such as saliva, where sample volume, abundance and integrity of mRNA are limited. The singleplex real-time PCR analysis following the preamplification can be performed with cost effective dye based chemistries in low reaction volumes with assay performance equal to conventional RT-qPCR. We show that the analysis can even be performed in 33 nanoliter reaction volumes on the BioTrove Open Array platform, which will enable high-throughput quantification in previously unparalleled extent, carrying out over 3000 individual qPCR reactions on a single slide.

In addition to the analytical targets, multiple transcripts for normalization can be integrated in the analysis. As these are reverse transcribed and amplified in the same reaction with the analytical targets, this also reduces the variability of the measurements to a large extent. Furthermore we present the use of several exogenous mRNAs as controls for sample analysis. These can be spiked into the sample prior to extraction or during the analytical process, thus controlling for inhibition, extraction efficiency and analytical variability. In the future they will also be used as semi-quantitative internal standards.

Finally our work indicates the necessity to incorporate appropriate carrier material such as tRNA into the handling of samples and reference material of low RNA concentration.

Overall, the new method allows the flexibility to establish extensive expression patterns of well over 30 targets for all kinds of biological samples and RNA concentrations. The high specificity and the implementation of extensive controls present a major advancement for research and clinical analysis of nucleic acids by qPCR and also other downstream methods.

Successful measurement of gene expression by quantitative PCR and DNA chip analysis with RNA derived of FFPE material.

Sybille Matthey1, Vlad Popovici2, Janine Antonov1, Andrea Oberli1, Anna Baltzer1, Mauro Delorenzi2 and Hans Jörg Altermatt3 and Rolf Jaggi1

1Department of Clinical Research, University of Bern, CH-3010 Bern, Switzerland, 2Swiss Institute of Bioinformatics (SIB), CH-1015 Lausanne, Switzerland and 3Pathology Länggasse, CH-3012 Bern, Switzerland

Email: rolf.jaggi@dkf.unibe.ch

Expression profiling with DNA chips is very popular and widely used in several areas of research including breast cancer. Until now, this technology depended on intact or at least good quality RNA which can only be isolated from fresh or freshly preserved tumor material (e.g. by snap freezing tumor material). Such material is not collected on a regular basis and therefore, only exists in very limited sets of samples. On the other hand, many thousands of samples exist as Formalin-fixed, Paraffin-embedded (FFPE) blocks, as they are collected and preserved in routine diagnostics. Even more interesting are samples, which were collected in the context of clinically controlled studies. Unfortunately, formalin fixation and paraffin embedding does not only lead to partial degradation of RNA, it also leads to extensive chemical cross-linking between nucleic acids (intra- and intermolecular) and between nucleic acids and proteins. As a result, RNA can only be isolated from tissue homogenates after extensive digestion with proteinase. We developed a special de-modification procedure which reverts N-methylol bonds formed during fixation between nucleotide bases and formaldehyde and which hydrolyzes methylene bridges between nucleic acids. The resulting RNA is highly suited for down-stream applications, e.g. TaqMan expression assays when used in combination with gene-specific cDNA and optimized TaqMan assays. The same RNA was also used for DNA chip analyses: RNA was amplified in a single round of random primed, T7-tagged reverse transcription followed by in vitro transcription in the presence of biotinylated or Cy3-labeled nucleotides. The resulting cRNA was hybridized to 12k CombiMatrix arrays and 44k Agilent arrays. CombiMatrix uses 35-40-mer oligonucleotides synthesized in a special porous layer, Agilent uses 60-70-mer oligonucleotides synthesized on glass slides. The technical quality of chips was assessed by comparing the same probe on different arrays (technical replicates). More interestingly, expression values from FFPE-derived RNA were compared to expression values from intact RNA (derived of snap frozen material of the same tumors). Correlations between such matched samples were in the range of 0.80-0.95, depending on the gene list of interest. We also compared DNA chip with QPCR for a number of genes: again, good correlations were observed between QPCR and DNA chips for both, good quality RNA (snap frozen) and FFPE-derived RNA.

We have developed a rapid and simple procedure for RNA isolation from FFPE material. The resulting RNA is suitable for QPCR and DNA chip based analyses. Samples from a clinical study on the efficacy of Tamoxifen and Letrozole for which only FFPE blocks exist, are currently being analyzed.

Simple & effective measures to increase consistency from sample to Ct.

Kavanagh I.

Thermo Fisher Scientific, ABgene, Great Britain (United Kingdom)

Email: ian.kavanagh@thermofisher.com

There are many choices a user must make in order to establish a viable QRT-PCR procedure. At Thermo Scientific (ABgene) we have identified aspects within a QRT-PCR protocol, ranging from sample preparation through to amplification, which can introduce variability to data. Introduction of simple, but effective measures can result in increased consistency throughout QRT-PCR.

During sample preparation contamination of an RNA sample with genomic DNA can lead to decreased reaction efficiency and false positives. DNase I is commonly used for removing DNA contamination, but this method is both time-consuming and increases the risk of RNA degradation. This degradation can occur during the harsh inactivation conditions of the DNase I. We have developed an alternative method that degrades contaminating DNA, which is equal in performance to DNase I, but also saves time and effort.

A choice of priming strategy must be made to initiate reverse transcription (RT). We have assessed the effect that a variety of alternative RT priming methods can have on the sensitivity of QPCR in different applications. We found that using a gene-specific primer for RT demonstrated the most sensitive QPCR amplification, with earlier Ct values. However, gene-specific priming limits the number of cDNA pool applications. We found that a blend of anchored oligo-dT and random hexamers generated the best Ct and end-point readings. In addition, this blend allows greater flexibility in the choice of endogenous control genes.

During the amplification step of QRT-PCR, dUTP is normally used in conjunction with the enzyme Uracil-N-Glycosylase (UNG) in order to remove any amplicon carry-over contamination. When using dUTP, higher Ct values and decreased QPCR reaction efficiencies of up to 5% are generated. Therefore a further way to increase consistency is to use a master mix that contains dTTP instead of dUTP.

Thermo Scientific has discovered that the use of white polypropylene plastic consumables can eliminate inefficient signal reflection observed in natural (clear) or frosted polypropylene. Different shades of white were tested to determine the optimum colour to provide the most sensitive detection. Despite the benefit of generating more reliable data, one of the biggest problems when using white plates has been visualizing the master mix in the well. One of our recently launched products, ABsoluteTM Blue QPCR master mix, solves this problem. This QPCR master mix contains an inert blue dye to significantly enhance the contrast between reagent and plastic and make verification of master mix dispensing quick, easy and foolproof.

The most important aspect of QPCR is increasing the consistency and reproducibility of the results generated. By employing a number of simple yet effective measures, users can significantly reduce many of the variables inherent in the QPCR process.

Pre-amplification with standardized mixtures of internal standards enables highly multiplexed Quantitative PCR analysis when sample size is limited or restricted by the volume requirements of nanofluidic systems.

James C. Willey1, Erin, L. Crawford1, Charles Knight2, Bradley Austermiller2

1University of Toledo, Toledo, Ohio, United States of America and 2Gene Express, Inc. Toledo, Ohio, United States of America

Email: james.willey2@utoledo.edu

There is increasing need to quantify transcript abundance of many genes in small samples such as single cell samples in stem cell differentiation experiments, or needle biopsy or microdissected samples in diagnostics. In addition, in nanofluidic systems input sample volume may be limited to the point where signal is lost. Signal loss occurs because no transcript molecules are present, not because the method is limited with respect to signal amplification. One way to address this is sample pre-amplification. Pre-amplification may increase the number of transcript molecules representing a particular gene by many orders of magnitude. However, if the inter-transcript variation in PCR amplification is not controlled, two rounds of amplification may increase analytical variation in relative representation of the transcripts representing different genes. In this study, Standardized RT (StaRT)-PCR was used in two rounds of amplification of 96 genes in the Stratagene Universal Reference RNA. In the first round, cDNA was mixed with a standardized mixture of internal standards (SMIS) containing an internal standard for each of the 96 genes and primers for each of the genes and PCR amplified for 35 cycles. Next, products from round one were diluted, aliquoted, and each aliquot was combined with primers for one gene, and amplified for an additional 35 cycles. No additional cDNA or SMIS was added. Expression values obtained from this method were highly correlated with values obtained by typical one round amplification (R = 0.993, p < 0.001). Products from round one could be diluted as much as 100,000-fold and still be quantified following round two amplification. Thus, using this method 96 genes were measured in the same amount of cDNA typically used to measure one gene with one round of PCR. This method was then successfully implemented in the high throughput Standardized Expression Measurement (SEM) Center and applied to quantify transcript abundance in a series of transthoracic fine needle aspirate biopsies from suspected lung cancers. This method should enable wider application of nanofluidic systems to quantify transcript abundance by quantitative RT-PCR and increase the number of clinical samples that may be assessed. J.C. Willey has equity interest in and is a consultant for Gene Express, Inc. E.L. Crawford has equity interest in Gene Express, Inc.


Session:   qPCR BioStatistics & BioInformatics

Chair:   M. Pfaffl / T. Bar

Lecture hall   HS 15

10 years of qPCR Data analysis, Biostatistics and Bioinformatics. Recent advances and new perspectives.

Michael W. Pfaffl

Lehrstuhl fuer Physiologie; Physiology - TU München, Germany

Email: michael.pfaffl@wzw.tum.de

Quantitative real-time RT-PCR (qRT-PCR) is widely and increasingly used in any kind of mRNA quantification, because of its high specificity, extreme sensitivity, reliable reproducibility, and wide dynamic quantification range. While qRT-PCR has a tremendous potential for analytical and quantitative applications, a comprehensive understanding of its underlying principles is important. Beside the classical RT-PCR parameters, e.g. primer design, RNA quality, RT and polymerase performances, the fidelity of the quantification process is highly dependent on a valid data analysis.

This review talk covers all aspects of data analysis in real-time PCR: data acquisition and threshold cycle determination procedures, e.g. trueness, reproducibility, and robustness of fluorescence readout; the potentials of data modification by the cycler software, e.g. curve smoothing, background correction, data adjustment/ normalization, and generation of standard curves. The talk will focus particularly on relative quantification methods using efficiency corrected quantification models. Furthermore useful and new bioinformatical, biostatical as well as multi-dimensional expression software tools will be presented.

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. However, inhibition of PCR is common when working with biological samples and may invalidate this assumption. Herein we present the Outlier function of Kineret, software for real-time PCR data analysis, management and validation. Outlier consists of two parts, kinetics outlier detection and CT-outlier detection. The kinetics outlier detection is based on multivariate analysis of geometric properties of amplification curves. Testing this function, we could identify 80-100% of artificially inhibited samples with significantly aberrant kinetics. The alteration of the kinetics was associated with a shift of about half cycle. The CT-outlier detection function utilizes the predefined experimental design, including definition of replicates, to detect PCR with outlying CTs. Intersecting the findings of the two methods results in robust validation tool for real-time PCR that enables evidence-based decision making in research and diagnostics.

Advanced and universally applicable models for relative quantification with flexible inter-run calibration and proper error propagation.

Hellemans Jan, Vandesompele Jo

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium

Email: Jan.Hellemans@UGent.be

Since its introduction more than 10 years ago, qPCR has become the standard method for quantification of nucleic acid sequences. To address the shortcomings of the available quantification strategies (and software tools), we developed a quantification framework based on multiple reference gene normalization, inter-run calibration and proper error propagation along the entire calculation track.

Two different experimental set-ups can be followed in a qPCR relative quantification experiment: sample maximization (recommended approach) or gene maximization (to be used in prospective studies). Whatever set-up is used, inter-run calibration is required to correct for possible run-to-run variation whenever not all samples for a given gene are analyzed in the same run. To this purpose, the experimenter needs to analyze so-called inter-run calibrators (identical samples that are tested in both runs). By measuring the difference in quantification cycle or normalized relative quantity (NRQ) between the inter-run calibrators in both runs, it is possible to calculate a calibration factor to remove the run-to-run difference. Inter-run calibration based on NRQ's has the important advantage that independently prepared cDNA of the same RNA source (or a biological replicate) can be used as a calibrator in the different runs. In contrast, calibration based on Ct values should be used if the inter-run calibrators can not be normalized (e.g. plasmid DNA). We present advanced algorithms, using imputation of missing data, to allow inter-run calibration for all types of experimental design.

Relative quantification using the delta-delta-Ct formula requires the selection of a reference sample (or so-called within-run calibrator). In principle any sample, or even an arbitrary cycle value can be chosen as a reference. The choice of reference cycle value does not influence the relative quantification result, but profoundly affects the final error on the relative quantities if the error on the estimated amplification efficiency is propagated in the calculations. We demonstrate that the overall final error can be minimized by selecting the average Ct value of all samples for a given gene as a reference for that gene.

The proposed calculation framework improves on the existing quantification strategies to provide more reliable results, and a better estimate of their error. To address the problem of the vast number of calculations, we partially implemented this framework in the existing qBase software [1]. The complete framework will soon be available in Biogazelle's platform independent and powerful qBasePlus software, providing an intuitive user interface for streamlined processing of all kinds of qPCR data.

Real-time PCR Expression Profiling.

Kubista Mikael

TATAA Biocenter, Sweden

Email: mikael.kubista@tataa.com

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 proper experimental design is required as well as methods to extract information from the measured data. In my talk I will present some of the first real-time PCR expression profiling studies, starting from the collection and pre-processing of real-time PCR data, to the classification of samples as well as genes with some of the most powerful methods available (www.multid.se). These approaches, which still are in their infancy, are expected to become the golden tool in future drug discovery, molecular diagnostics and prognostics, as well as in basic research in molecular cell biology and molecular physiology.

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

A novel approach, based on in silico analysis of plant transcriptome and web search, for the rapid identification of candidate reference genes for gene expression studies.

Valeria Terzi1, Gian Paolo Ciceri1, Paolo Provero2,Caterina Morcia1, Primetta Faccioli1.

1C.R.A., Istituto Sperimentale per la Cerealicoltura, Via S. Protaso 302, I-29017 Fiorenzuola d’Arda (PC), Italy and 2Dipartimento di Genetica, Biologia e Biochimica, Università di Torino, Via Santena 5bis, I-10100 Torino, Italy

Email: v.terzi@iol.it

Advanced gene expression analysis methods, such as microarray and Real Time PCR, as well as more traditional ones, such as Northern blotting, require efficient normalization approaches to be really informative. Therefore, expression results are usually normalized against a control gene that should be expressed in an unchanging fashion regardless of experimental conditions (i.e. tissue types, developmental stages, sample treatments). However the utilization of single housekeepers can’t assure a not-biased result: as a consequence new normalization methods employing multiple housekeeping genes and normalizing using their mean expression have been proposed, but finding a good set of reference genes is for sure a non trivial problem requiring quite a lot of lab-based experimental testing. In this work (Faccioli et al, DOI 10.1007/s11103-006-9116-9) , a novel, agile approach, based on in silico analysis of plant transcriptome, has been developed, for the rapid identification of candidate reference genes to be used as endogenous control in quantitative gene expression analysis. The fundamentals of the analysis is based on the fact that housekeeping genes are expressed across a variety of tissues and biological conditions, as a consequence if we select those transcripts that are present in a remarkable number of different cDNA libraries we should be able to more easily identify potentially constitutive genes to be used as reference genes in normalization of expression data. The TIGR Gene Indices (http://www.tigr.org/tdb/tgi/), examples of public EST-based species-specific databases, have been used to provide such information. Moreover, a web search engine optimization has been set up and applied to collect the most interesting information on the expression profiling of candidate housekeepers on specific experimental conditions. As a validation test for the method, a qPCR analysis has been carried out to verify the expression profile of the selected genes in different barley organs and developmental stages and on stress conditions.

CAmpER - A software for the calculation of amplification efficiencies for real-time PCR-experiments.

Blom J., Rückert, C., Kalinowski, J., Goesmann, A.

Center for Biotechnology, Bielefeld University, Germany

Email: jblom@cebitec.uni-bielefeld.de

Quantitative real-time PCR has become one of the fundamental methods of modern molecular biology and medicine for the measurement of gene expression. For the analysis of a real-time PCR experiment it is crucial to have exact knowledge of the underlying PCR kinetics. Especially the amplification efficiencies of an experiment are of importance for the analysis of gene expression ratios. For this reason the software CAmpER was designed as a software-tool for the automatic analysis, annotation, and storage of real-time PCR experiments performed with different real-time PCR systems, currently the Lightcycler® (Roche Diagnostics) and the Opticon® (Bio-Rad Laboratories, Inc.).

CAmpER obtains the experiment data by automatically importing the fluorescence and melting-curve data from the output files of the real-time PCR-system. Based on the imported fluorescence data the amplification efficiencies are calculated for single samples by two different independent algorithms. The first algorithm is based on the DART-PCR method1, the second algorithm is based on a four parametric logistic model2 of the fluorescence curve. Both algorithms were revised and improved, especially the DART-algorithm, now calculating precise crossing points, which were integer in the original version.

The calculated amplification efficiencies are used to calculate efficiency-corrected crossing points. These corrected crossing points can be used to estimate gene expression ratios for all samples, which are more accurate than gene expression ratios calculated from uncorrected crossing points.

Another function of CAmpER is the consistent storage of all relevant information of a real-time PCR experiment, giving CAmpER a basic LIMS functionality. It allows users to annotate the samples used in the experiment as well as the experimental parameters in a standardized format. The annotation information and all other experiment data is stored using a relational database management system (here MySQL). The modular design of CAmpER allows for extension of the software, for example to support additional real-time PCR systems in the future.

To ensure easy access to stored data and easy use of the implemented functions a convenient user interface was designed. This user interface is implemented as a web-based frontend, making a local installation of CAmpER needless.

Session:   RDML discussion forum

Chair:   J. Hellemans

Lecture hall   HS 15

RDML: real-time PCR data markup language.

Vandesompele Jo, Hellemans Jan

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium

Email: joke.vandesompele@ugent.be

1. Introduction

It is currently very difficult to share qPCR data between different laboratories, or exchange data between different software packages or analysis tools. The problem is founded in the data collection software packages that, depending on the instrument company, export information in various file formats (.csv, .txt, .xls), with different layout and data field terminology. A common universal format would allow easy exchange of raw annotated data and analysis settings for qPCR experiments. This would make it possible to include qPCR data and analysis settings in scientific papers, allowing both reviewers and readers to re-analyse the data, much like the MIAME guidelines propose for microarray experiments. In principle, the universal data format should contain sufficient information to understand the experimental setup, re-analyse the data and interpret the results. This data format could be part of yet to be established MIARE guidelines (Minimal Information About Real-time Experiments).

2. Discussion

We have drafted a proposal for a universal data format for the exchange of real-time PCR data, termed RDML (Real-time PCR Data Markup Language - http://medgen.ugent.be/rdml/). We propose to use an XML-based file type because this format is independent of computer hardware, operating system or available software package, and can be extended in the future to include additional information if required. The aim of this session is to discuss and finalize the RDML specifications. Points to discuss include:

1. What information must be included?

2. Which fields are required and which are optional?

3. Which fields are free text, and which have predefined values?

4. What terminology should be used?

5. How to obtain broad acceptance of this standard?

6. Is there a need for an RDML working group to continue the development of RDML?


Session:  New Diagnostic applications with qPCR

Chair:        S. Bustin / K. Stanley

Lecture hall   HS 14

Analysis of expression signatures associated with microvascular invasion in colorectal cancer.

Rebecca E Hands 1, Keith Stanley 2, Sina Dorudi 1, Stephen A Bustin 1

(1) Queen Mary University of London, United Kingdom (2) AusDiagnostics, Sydney, Australia

Email: s.a.bustin@qmul.ac.uk

The development of molecular analytical techniques and their application to clinical diagnostic studies has been rapid. However, the reliable interpretation of results obtained from gene expression analyses is critically influenced by the selection of appropriate study material. Colorectal cancer biopsies contain multiple cell types, are multiclonal and are characterised by distinctive histopathologies with discrete genetic and gene expression profiles. As a result, gene expression profiles from bulk biopsies are likely to obscure clinically relevant expression signatures from areas associated with poor treatment outcome, such as those characterised by vascular and lymphocytic infiltration. A critical consequence of this heterogeneity is that such samples are unable to provide robust expression signatures capable of accurate prognostic stratification of individual colorectal cancer patients following surgery. Our solution involves the establishment of a tumour atlas for clinically relevant histopathological areas in colorectal cancers. We achieve this by using laser capture microdissection in combination with multiple tandem RT-qPCR to delineate distinct gene expression features associated with high metastatic potential and poor treatment outcome.

Use of Tomato Mosaic Virus (ToMV) as Internal Positive Control (IPC) in different RT-PCR settings.

Helga Mairhofer1, Martin Obermeier1, Günter Adam2, Hans Nitschko1

(1) Max von Pettenkofer-Institute, Ludwig-Maximilians-University Munich, Department of Virology, Munich, Germany (2) Pflanzenschutzamt Hamburg, Biozentrum Klein Flottbek, University of Hamburg, Hamburg, Germany

Email: nitschko@mvp.uni-muenchen.de

Objectives: The use of an Internal Positive Control (IPC) in each individual specimen is highly recommended and increasingly practiced in routine clinical virus diagnostics. We used Tomato Mosaic Virus (ToMV), a highly stable single-stranded plant RNA virus, to monitor the efficacy of nucleic acid isolation, reverse transcription and amplification in different RT-PCR settings.

Methods: A short 190 nt sequence located in the coat protein region was coamplified with ToMV- specific primers after addition of ToMV-IPC directly into the clinical specimen prior to extraction.. We used different nucleic acid isolation systems to extract viral and IPC RNA: Roche, MagNAPure, Roche AmpliPrep and the High Pure Viral Nucleic Acid Isolation Kit as a manual extraction protocol. The isolated RNAs were investigated using different amplification and detection platforms: Roche LightCycler 2.0, ABI TaqMan 7500 fast and conventional RT-PCR with visualisation of the amplicons by agarose gel electrophoresis. The concentration of the added ToMV was carefully adjusted to ensure reliable and reproducible detection without significant loss of sensitivity especially for weak positive RNA signals from respective viruses to be detected in the patient specimen. We optimized the protocol by evaluation different primer/probe concentrations for reverse transcription and RT-PCR amplification using a variety of difficult clinical specimen such as stool, BAL, biopsies, pharyngeal lavages, swabs, urine and ENTA but also peripheral leucocytes, plasma and serum. Norovirus positive stool samples and influenza A virus (H5N1) positive materials were applied to analyze in detail the performance of the spiked, co-amplified IPC as well as the amplification of the specific viral RNAs.

Results: More than 300 different clinical specimen were analysed. The IPC could be reliably and repeatably detected in most of the samples. In some specimen inhibition was clearly demonstrated as indenpendently of the extraction/amplification protocol no or only weak IPC signals were detectable resulting in a shift of Ct-values and/or flattened amplification curves. Upon ten-fold dilution IPC and virus specific RNA-signals could be recovered with appropriate signal intensity. We could also identify samples with insufficient efficacies in either RNA extraction, reverse transcription and/or amplification (below 5% of all samples), clearly depending on the type of sample material, extraction method and amplification conditions. As expected not all isolation/amplification methods are equally well suited to guarantee optimal performance.

Conclusions: Tomato Mosaic Virus is a stable, reliable and easy to handle plant virus which can be isolated in high concentrations, stored unendangered and used safely as an Internal Positive Control in RT-PCR detection protocols for human RNA viruses in clinical specimen.

Evaluation of endogenous control genes for real-time quantitative PCR in breast cancer tissues.

McNeill R.E., Miller, N. and Kerin, M.J.

Department of Surgery, Clinical Science Institute, National University of Ireland, Galway, Ireland

Email: roisin.mcneill@nuigalway.ie

Quantitative real-time PCR (qPCR), one of the most sensitive and specific quantitative methods for gene expression analysis has become the basis of many breast cancer biomarker studies as well as novel diagnostic and prognostic assays. Normalisation of relative qPCR data is required to control for variation introduced during sample preparation, particularly to control for differences in the quantity and quality of RNA used in each reaction. Endogenous control (EC) genes, used to normalise relative qPCR data, should ideally be expressed constitutively and uniformly across treatments in all test samples. The aim of this study was to identify the most suitable endogenous control gene(s) from a panel of twelve candidates ( GAPDH , ACTB , TFRC , ABL , PPIA , HPRT , RPLP0 , B2M , GUSB , MRPL19 , PUM1 and PSMC4 ) for the quantification of gene expression by relative comparative qPCR in breast cancer tissues using the oestrogen receptor alpha ( ESR1 ) transcript as a target gene.

Primary breast tumour tissues, malignant (n=21) and benign (n=8) were obtained from consenting patients during primary curative resection in University College Hospital, Galway. Normal tissues from patients undergoing reduction mastopexy were used as calibrators (n=2). Following RNA isolation and cDNA synthesis, gene expression was measured by qPCR using TaqMan® Endogenous Control Assays or TaqMan® Gene Expression Assays and the ABI Prism® 7000 Sequence Detection System. PCR amplification efficiencies for each assay were calculated using standard procedures. Expression variability was analysed using geNorm1 and Normfinder2. Bartlett’s test (Minitab v.14) was used to compare both the pooled variances within tissue type for each EC and the variability of normalised ESR1 expression using different ECs.

There was a significant difference in candidate EC variability within group (P<0.01). GeNorm and Normfinder identified the same two genes as most stable. GAPDH was less stable than either of the two genes identified. ESR1 expression was estimated to be appreciably higher in malignant tissues than in benign tissues irrespective of which EC was used. In conclusion, two genes have been identified as good candidate ECs for the normalisation of qPCR gene expression data in these tissues.

1. Vandesompele, J.; De Preter, K.; Pattyn, F.; Poppe, B.; Van Roy, N.; De Paepe, A.; Speleman, F. Genome Biol 2002, 3, (7), RESEARCH0034.

2. Andersen, C. L.; Jensen, J. L.; Orntoft, T. F. Cancer Res 2004, 64, (15), 5245-50

Fluorogenic Quantitative PCR for Non-laboratory Applications.

Lee M.1, Squirrell D.1, and Wakeley, P.2

1Enigma Diagnostics Ltd, Building 224, Tetricus Science Park, Dstl Porton Down, Salisbury, Wiltshire SP4 0JQ and 2Veterinary Laboratory Agency, Technology Transfer Unit, Biotechnology, New Haw, Addlestone, Surrey KT15 3NB

Email: martin.lee@enigmadiagnostics.com

For several years both defence and homeland security organisations have used QPCR as a powerful tool for the detection of bacterial and viral biological agents. Field laboratories using commercial thermal cyclers and nucleic acid extraction systems have been deployed, but skilled operators and an ensemble of consumables were required. Enigma Diagnostics has developed a better solution. The Enigma FL is a portable device that can be operated after ten minutes of training. It carries out automated extraction, amplification and analysis with the sample loaded into a single cartridge containing all of the reagents, including a freeze-dried PCR mix. The device can be operated from mains, battery or a vehicular power supply opening up numerous new applications for nucleic acid testing outside of the laboratory. Here we present a detailed technical overview of the instrument and technology within, whilst illustrating the utility of the device for the veterinary testing for a reportable infectious disease, bovine viral diarrhoea virus (BVDV), in a UK-based field trial.

Real-time PCR in Diagnostic Microbiology - a review on 9 years of R&D in an academic environment.

Reischl U.

University Hospital of Regensburg, Regensburg, Germany

Email: udo.reischl@klinik.uni-r.de

For more than a century, pathogenic microbial detection and identification have relied on the ability to cultivate organisms in the diagnostic laboratory. About twenty years ago a practical method of amplifying DNA for the clinical laboratory appeared - well known today as the PCR. Application of nucleic acid amplification, distinguished by its high analytical sensitivity and specificity, circumvent the necessity for microorganisms to be isolated in pure culture prior to their definitive identification. In the early 90's many of us played around with block cycler PCR devices, special brands of thermostable polymerases and semiquantitative amplicon detection in agarose gel analysis. But the true quantum leap in technology was associated with the event of real-time PCR - where detection of amplicons and monitoring of the amplification process was possible after each thermocycle. With the introduction of the LightCycler instrument in August 1998, the advantages of real-time PCR were accessible even for routine laboratories and molecular diagnostics become particularly popular. Combining PCR amplification and probe detection of target nucleic acids in the same closed reaction vessel permits dynamic assessment of PCR amplicons, quantification of target organisms and concurrently minimizes the possibility of contamination by exogeneous nucleic acid in the workflow of molecular microbiology. Due to the widespread application of real-time PCR and the development of various assay formats, a huge spectrum of diagnostic in-house protocols or commercial kits are now available to detect almost any bacterial or fungal organsim as well as related pathogenicity factors. The versatility of this approach is exemplified by applications which identify human pathogens, relevant mutations or antimicrobial resistance genes. Moreover, a number of automated nucleic acid extraction systems will be discussed which have been developed to standardize and economize the diagnostic workflow from receipt of a clinical specimen to reporting the corresponding PCR result. Although the present technology can be considered to be robust and reliable for routine purposes, further developments are ahead - and high resolution melting, multiplexing or high-throughput testing in microwell formats will definitely expand the spectrum of diagnostic applications in the months or years to come.

Multiplex quantitative PCR for detection of Ehrlichia canis, Babesia canis and canine ACTB gene.

Ofer Peleg1, Gad Baneth2, and Shimon Harrus2

1Zotal LTD, Israel and 2School of Veterinary Medicine, Hebrew University of Jerusalem

Email: oferp@zotal.co.il

The ricketsia Ehrlichia canis and the protozoon Babesia canis vogeli share the Ixodid tick Rhipicephalus sanguineus as a vector. Canine monocytic ehrlichiosis is acknowledged as an important and potentially fatal infectious disease of dogs and other members of the Canidae family, while parasitic Babesia species infect erythrocytes, causing anemia in the host. As a result of their common vector, concurrent infections with both pathogens are common. Therefore, in this study we attempted to construct a multiplex quantitative PCR assay for the detection of both pathogens simultaneously.

Using the qPCR Singleplexer and Multiplexer software (GenAphora) for singleplex and multiplex primer and probe design, Singleplex qPCR assays for detection of the E. canis-16S rRNA gene, Babesia vogeli-hsp70 gene and the canine beta-Actin (ACTB) as a reference gene were constructed. In addition, a multiplex assay for the simultaneous detection of all three genes in a single tube was constructed. The sensitivity of the multiplex was reduced compared to the singleplex assays. The singleplex assays could demonstrate detection of 1-10 molecules of organism (DNA copies)/µl. while the multiplex assays were able to detect ~100 molecules of organism (DNA copies)/µl. The probable causes for this reduction are system resources deficiency (e.g. nucleotide and ion deficiency) due to the occurrence of 3 simultaneous reactions, and the presence of 3 different primer probe-sets in the same tube in addition to the presence of genomic-DNA and blood DNA extraction byproducts in the reaction tube. Of these, the major contributor for the multiplex sensitivity reduction was the existence of several reactions in the same tube. Improvement in the sensitivities of the multiplex test is currently underway.

The present assay enables the quantitative detection of two common canine blood pathogens simultaneously. It can thus indicate the institution of proper treatment and monitoring the response to treatment with the changes in pathogen numbers in the blood.

Rapid and sensitive detection of invasive fungal infections by a 2-step pan-fungal real-time PCR assay.

S. Preuner 1, T. Lion 2

Children's Cancer Research Institute, Vienna, Austria

Email: sandra.preuner@ccri.at

Rapid and sensitive detection of invasive fungal infections

by a 2-step pan-fungal real-time PCR assay

S. Preuner, T. Lion, Children´s Cancer Research Institute, Vienna, Austria

Invasive fungal infections (IFI) are a major cause of life-threatening situations in immunocompromised patients (bone marrow transplant patients, patients with haematological malignancies, HIV patients). The incidence of IFI in these patients is estimated at 10-40% . The most common organisms involved are Aspergillus and Candida species (> 90%), with a mortality rate of 10-40%. The introduction of new antifungal and antineoplastic treatment results in changes in the epidemiology of fungal infections. The so-called “emerging fungi” including e.g. Fusarium spp., Mallassezia spp., Trichosporon spp., Cryptococcus spp. play an increasing role. Thus early, sensitive and broad detection of fungi has to be implemented. Currently used detection methods, such as microbiological cultivation, serological tests, histological techniques or imaging methods are mostly inconsistent in sensitivity and specificity or are time consuming. Therefore antifungal treatment is often empirical, resulting in unnecessary treatment of patients in absence of IFI or in delay of treatment in the presence of IFI. To provide a reliable tool for diagnosis of IFI, we have developed a two-step pan-fungal real-time assay targeting the 28s rRNA gene. Assay I (“Mould-Assay”) covers all clinically relevant Aspergillus species and a number of other important moulds. Assay II (“Yeast Assay”) detects all Candida species, Trichosporon spp., Cryptococcus spp, and a number of filamentous fungi not covered by the Mould Assay. In total, more than 80 different fungal species, including also a large panel of “emerging fungi” can be detected and quantified. High homology with human 28S rRNA gene required the employment of LNA (locked nucleid acid) modified primers and probes, resulting in enhanced affinity for the complementary sequence. Due to the composition of the cell wall, DNA extraction had to be modified by the introduction of enzymatic and mechanical pre-treatment.

The occurrence of contaminations by airborne spores in reagents for DNA extraction and PCR were a major issue. This problem was overcome by using molecular grade reagents and the application of a closed extraction system, the MagnaPure System (Roche Diagnostics, Switzerland). The detection limit of the assay is 1 fg fungal DNA. In clinical samples, 10 organisms per ml peripheral blood/plasma can be detected. The 2-step pan-fungal real-time PCR assay is a powerful tool for early, sensitive and broad-spectrum diagnosis of IFI, permitting timely onset of antifungal treatment based on firm evidence.

Rapid, On-demand Detection of Drug Resistant Microorganisms by using the GeneXpert.

Persing DH.

Cepheid, United States of America

Email: david.persing@cepheid.com

Detection of drug-resistant organisms, especially those that cause hospital-acquired infections, has become an increasingly important role for the clinical laboratory. Conventional culture methods are too slow to be of use in the most effective hospital infection control strategies, which require rapid recognition and cohort isolation. Molecular methods based on PCR have improved turnaround time, but their dependence on batch-mode processing can stretch effective turnaround times to 24 hours or more. We have developed rapid cartridge based tests for several drug resistant organisms. These tests carry out automated sample extraction from crude clinical specimens such as sputum, blood, or swabs and use realtime PCR to detect targets and internal process controls that are multiplexed in 4 to 6 color channels. Results are delivered in as little as 30 minutes in order to facilitate high-impact medical management or treatment decisions. Because each cartridge contains it own controls, tests can be run on-demand, in batches or one at a time, within the random-access GeneXpert system. Tests for different analytes, using diferent reagents and distinct thermal cycling conditions, can be run side-by side upon arrival in the lab, thus keeping effective turnaround time to a minimum. Data will be presented from Cepheid's test released for detection of methicillin resistant S. aureus, and for tests in development for drug resistant tuberculosis and influenza virus.

Applications of high resolution melt curve analysis for genetic diagnostics.

White H.1, Watkins G. 1, Hall V.1 and Cross NCP. 1

(1) National Genetics Reference Laboratory (Wessex), United Kingdom

Email: hew@soton.ac.uk

High resolution melt curve analysis (HRM) is a simple & cost effective post-PCR technique that can be used for applications such as high throughput mutation scanning, detection of somatic mutations, genotyping & methylation profiling. The technique requires the use of standard PCR reagents only and a dsDNA binding dye. NGRL (Wessex) have evaluated several applications of HRM for use in genetic diagnostics.

We have evaluated the use of 3 machines for mutation scanning using HRM: RotorGene 6000 (Corbett Research), HR-1 and 384 well LightScanner (Idaho Technology). Eleven amplicons were analysed which varied in size from 139 - 449bp and had GC contents ranging from 22 - 79%. The types of mutations analysed included all possible point mutation base substitutions and 1 and 2bp insertions and deletions. 624 blinded samples were amplified in the presence of the saturating double stranded DNA binding dye LCGreen Plus (Idaho Technology) using a RotorGene 6000. Identical PCR products were analysed using HRM on the three platforms. The overall sensitivity and specificities for each machine were 100% & 95% (RotorGene 6000), 98.4% & 95% (HR-1) and 99% & 88% (LightScanner) respectively. We concluded that HRM is an extremely sensitive and specific technique for mutation scanning that could be easily integrated into clinical diagnostic pre-screening strategies. This closed tube method has advantages over current mutation scanning techniques since it requires no post-PCR handling (minimising the risk of PCR contamination) and no separation step, which improves analysis time. The technique can also be used to scan for somatic mutations and has a sensitivity of detection of approximately 2%.

NGRL (Wessex) has also investigated the use of HRM for methylation profiling and has compared the use of three dsDNA binding dyes: LCGreen Plus (Idaho Technology), Syto9 (Invitrogen) and EvaGreen (Quantace/Biotum). We have used the technique to set up a diagnostic screen for Prader Willi (PWS) and Angelmann syndromes (AS) which are two distinct neurodevelopmental disorders caused primarily by deficiency of specific parental contributions at an imprinted domain within the chromosomal region 15q11.2-13. Analysis of allelic methylation differences at this locus can discriminate the maternally and paternally inherited chromosome 15 and can be used as a diagnostic test for AS and PWS. HRM using LCGreen Plus, Eva Green and Syto9 was used to analyse methylation differences in an amplicon containing 21 CpG dinucleotides at the SNRPN locus in DNA samples from PWS (n=39), AS (n=31) and normal control samples (n=96). Results from the HRM assays were compared to those obtained using a methylation specific PCR (MS-PCR) protocol that is used commonly in diagnostic practice. Results from the HRM assays showed 100% concordance with the MS-PCR protocol and the methylation sensitive HRM assay using EvaGreen resulted in the most robust assay conditions with the lowest failure rate (0.6%).

qPCR analysis of molecular targets for developing world pathogen diagnosis; a multi-step approach to a multi-step problem.

Huggett J. F.

Centre for Infectious Diseases & International Health, University College London, United Kingdom

Email: j.huggett@ucl.ac.uk

Infectious diseases are a major cause of mortality and morbidity in the developing world. Tuberculosis (TB) causes ~2 million deaths per annum; this is effectively the death toll attributed to the 9/11 attacks on the World Trade Centre every 12 hours. Diseases like TB are notoriously difficult to diagnose. We are using qPCR and reverse transcriptase qPCR to establish the potential role of molecular methods in the management TB and Pneumocystis pneumonia (PCP), two respiratory infections with similar symptoms. While there are many reports describing molecular assessment using PCR for diseases like TB and PCP, the clinical utility of these tools has not been uniformly successful and they are generally not used as a routine diagnostic method, especially in the developing world.

There are many reasons why PCR has not been more successful in diagnosing TB and PCP, but a lack of consistency in the literature is a major factor. This is confounded by the frequently expressed notion that the publications have assessed the diagnostic efficacy of PCR. This notion is wrong. These reports are assessing the diagnostic efficacy of a multistep process, the final step of which is PCR. Consequently, for a report to be thorough there must be detailed explanation of chosen sample, storage and extraction procedure. Molecular assessment by PCR (or other enzymatic amplification methods) should also control for inhibition. Finally the PCR reaction used to detect the pathogen of interest should have a defined reproducibility, detection sensitivity and ideally efficiency.

We are approaching this problem by using in silico methods to assist the assay design and internal controls to provide confidence in our results. Concomitantly we consider different storage and extraction procedures from a range of clinical samples for optimal nucleic acid recovery and removal of inhibitors. We are also investigating the use of novel clinical samples including urine to target pathogen transrenal DNA as a tool for monitoring respiratory infection. Our infectious disease diagnostic and prognostic studies using real time PCR can provide valuable clinical information that will be important in the management of infectious diseases in the developing world.

Two new probes for Real-time PCR: EasyBeacons™ and HydrolEasy™ probes.

Christensen U.

PentaBase, Denmark

Email: ubc@pentabase.com

We would like to introduce two new probe systems for real-time detection during PCR amplification: EasyBeacons™ and HydrolEasy™ probes. Both type of probes are modified with the DNA analogue Intercalating Nucleic Acid (INA®) or INA®-like molecules and labelled with a fluorophore and a quencher. The fluorophores are compatible with commercially available real-time PCR instruments and hence the technology can be used on standard equipment.

The novel features of EasyBeacons™ and HydrolEasy™ probes are introduced by INA®. Common for the two probe systems are, that they are easy to design and very specific. INA®-modifications increase binding to their complementary targets with as much as up to 11°C per modification. They have an inherent quenching mechanism, yielding low background fluorescence from unbound probes. This quenching mechanism is independent of temperature, making multiplexing easier.

EasyBeacons™ are nuclease resistant probes that can be used both real-time during the amplification reaction and in end-point detections. This dual detection mechanism is making EasyBeacons™ ideal for detection of SNPs, MeC patterns and other variations, as the real-time result can be verified by an end-point affinity measurement, similar to the one used with non-specific dyes like SYBR® green. We will also present fast optimisation protocols for using EasyBeacons™. Examples shown will include the detection of methylation pattern of single CpG duplets as well as detection of challenging SNPs.

HydrolEasy™ probes are hydrolysed during the amplification reaction, cleaving the fluorophore from the rest of the probe. Besides being high affinty probes, due to the INA® modifications, the unbound probes have a low background fluorescence due to hydrophobic interactions between INA® modifications. HydrolEasy™ probes are ideal for two-step real-time PCRs.

TripleHyb real time PCR for detection of single nucleotide polymorphisms in the VEGF promoter region.

Susanne Füssel1, Susanne Unversucht1, Andrea Lohse1, Silke Tomasetti1, Anne-Katrin Rost2, Manfred P. Wirth1, Axel Meye1, Thomas Köhler2

1Dept. of Urology, Technical University of Dresden, Germany and 2AJ Roboscreen GmbH, Leipzig, Germany

Email: susanne.fuessel@uniklinikum-dresden.de

In this study the novel real time PCR detection format “TripleHyb” was optimized for the detection of single nucleotide polymorphisms (SNP) for the known C-460T polymorphism of the VEGF promoter. “T” instead of “C” at position -460 is believed to be associated with altered VEGF promoter activity possibly leading to increased incidence of different diseases and malignancies such as prostate cancer.

Briefly, the TripleHyb format besides a conventional set of primers comprises a pair of oligonucleotide probes which consist of a target-complementary subsequence and a target-unrelated subsequence for the formation of a stabilizing stem structure between the probes. The labeled upstream probe is cleaved by 5`-exonuclease function of taq polymerase as long as full complementary with target is achieved, whereas the probe is displaced if at least one mismatch is present resulting in lack of fluorescence signal. To find out at which position(s) of the upstream probe a mismatch causes SNP selectivity a common VEGF promoter specific set of primers and 5 different sets of either complete target matching or single mutated fluorescence labeled upstream and the corresponding non-labeled downstream probes were developed. The mismatches were located either at positions 1, 2, 3, 4, or 5 (probe set 1-5) of the upstream probe`s 5`-end. Optimizations of PCR conditions using a plasmid containing a wild-type or C460T polymorphism subsequence were performed. A sensitive detection of the specific genotype was feasible using probe sets 3, 4 and 5, respectively without need for running subsequent melting curve analysis. When applying a 1:1-mixture of both wildtype and mutated template variants a reproducible and reliable discrimination between the SNP variants was possible with all three systems. We conclude that with TripleHyb a broad hot spot region within the upstream probe may be exploited thus simplifying the probe design.

Set 3 was selected to analyze 80 DNA samples from prostate cancer patient for the respective mutation frequency. The clinical data are still in the evaluation process.

Allergen determination in food by multiplex qPCR.

Köppel R.

Kantonales Labor Zürich, Switzerland

Email: rene.koeppel@klzh.ch

The cantonal laboratory examines food samples for safety and fraud. Two to six percent of the human population exhibit allergic reactions due to the consumption of food containing allergens. 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 by the food law of Europe and Switzerland. In principle food samples need to be analysed for all these allergens. Although many allergen combinations are unlikely to be present in food, 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 determine contents of peanut, hazelnut, celery, soy and egg, milk, almond sesame, respectively. The Tests exhibit a good specificity and a sensitivity in the range of 0.01% to 10%. A comparison of ELISA-results with the PCR-results revealed a good qualitative accordance but a low correlation of the quantitative results.

Quantitative DNA Methylation Analysis.

Serena Vinci, Francesca Malentacchi, Roberta Cascella, Francesca Salvianti, Mario Pazzagli, Pamela Pinzani, Claudio Orlando.

Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, Italy

Email: c.orlando@dfc.unifi.it

Genome stability and normal gene expression are maintained by a constant and predetermined pattern of DNA methylation. ‘Epigenetics’ is defined as heritable changes in gene expression that do not result from alteration in the gene nucleotide sequence. These include DNA methylation and histone modifications, that provide an "extra" layer of transcriptional control. Epigenetic abnormalities have been found to be causative factors in cancer, genetic disorders and pediatric syndromes as well as contributing factors in autoimmune diseases and aging.

The detection of abnormal patterns of DNA methylation, beside the relevant insights in the comprehension of molecular mechanisms of these diseases, seems to provide a new and promising biomarker for identifying occult circulating DNA inside complex biological matrices (blood, urine, sputum, stool, etc.) for early cancer detection. It should be noted that changes in DNA methylation also occur in normal epithelia in physiological and pathological conditions. Thus, conventional qualitative techniques for methylated DNA sequence cannot guarantee the expected clinical relevance for routine applications. Quantitative Methylation Specific PCR (QMSP) is capable of detecting methylated alleles in the presence of 10000-fold excess of unmethylated alleles and in addition it can provide a quantitative estimation of the number of abnormally methylated sequences in clinical samples.

In our study QMSP was applied to determine aberrant promoter methylation profile of the 5’ region of 3 apoptosis-associated genes (Bcl2, hTERT e DAPK) in urine sediments as potential biomarkers for bladder cancer detection. DNA hypermethylation was detected in about 80% of bladder cancer patients, with a specificity of 91%. Methylation levels were variably but significantly associated with clinical and pathological markers.

Another interesting field of application of quantitative measurement of occult circulating DNA is represented by the non-invasive diagnosis of some pregnancy-associated diseases, such as pre-eclampsia. Up to now, the lack of a fetal DNA marker that can be universally detected in maternal plasma has limited the clinical application of this technology. It is now evident that epigenetic differences between the placenta and maternal blood cells could be used to detect fetal DNA in maternal plasma. Maspin gene promoter is heavily methylated in maternal blood cells and hypomethylated in the placenta. Placental-derived hypomethylated maspin is detectable in maternal plasma regardless of the fetal sex and genetic variations and thus could serve as a universal fetal DNA marker. Recently it was demonstrated that the promoter of the RASSF1A tumor suppressor gene is hypermethylated in the placenta but hypomethylated in maternal blood cells, a methylation pattern that is exactly opposite to maspin promoter. We explored both these strategies to set-up a fetal DNA marker easily detectable and quantifiable by real-time PCR.

Validation of StaRT-PCR for reliable and robust analysis of variably degraded formalin-fixed paraffin-embedded or fine needle aspirate biopsy samples.

James C. Willey1, Charles Knight2, Bradley Austermiller2, Thomas Blomquist1, Erin Crawford1, Elizabeth Peters2

(1) University of Toledo, United States of America (2) Gene Express, Inc. United States of America

Email: james.willey2@utoledo.edu

It is widely anticipated that a transcript abundance (TA) measurement method that generates data suitable for regulatory review will facilitate development of new drugs and improved diagnostic tests. A particular challenge is to maintain these performance characteristics using clinical samples that often unavoidably are degraded to different degrees. This study was an effort to assess the utility of Standardized RT-PCR (StaRT-PCR™) for reliable and robust analysis of formalin fixed paraffin embedded (FFPE) or transthoracic fine needle aspirate (FNA) biopsy samples. Seven genes were assessed relative to the ACTB loading control gene, including four genes reported to be associated with resistance to pemetrexed and three genes that accurately diagnose bronchogenic carcinoma, MYC, E2F1, and CDKN1A. A 3’, middle, and 5’ region of the transcript was assessed for each gene. Each region was selected for analysis based on thermodynamic analysis to identify short sequence regions most resistant to degradation. Cell culture populations from each of seven carcinoma cell lines were split into two samples, one of which was pelleted and snap frozen (FF), the other pelleted, formalin fixed, and paraffin embedded (FFPE). RNA was extracted from each sample, DNase treated, quantified, and reverse transcribed using multiple different RT priming methods. Genomic DNA was assessed and ensured to be negligible. TA for each gene was measured relative to a known quantity of its respective internal standard within a standardized mixture of internal standards (SMIS<sup>TM</sub>). This provided the performance characteristics recommended by the FDA to generate data suitable for regulatory review, including intrinsic quality control to prevent false negatives and false positives and to control for effects of interfering substances such as gene-specific inhibitors. The integrity of each RNA sample was assessed according to yield of ACTB cDNA molecules/ng RNA. For the seven FFPE samples, the ACTB cDNA yield ranged from 0.3% to 1.4% of that obtained from the respective matched FF sample. In spite of extreme degradation, TA value for each target gene normalized to ACTB was highly correlated for each FF/FFPE pair. However, the correlation was least for the two FFPE samples with the lowest ACTB cDNA yield from RT, each with 420 ACTB molecules/ng RNA. Primers spanning the most 5’region of each transcript yielded results best correlated between FF and FFPE. Spontaneous priming was as efficient as oligodT or gene specific priming and also yielded reliable FF/FFPE results. Based on this study, FFPE or other clinical samples will be judged suitable for analysis if RT yields more than 1,000 ACTB cDNA molecules/ng RNA. The StaRT-PCR reagents validated in this study will be used in clinical trials. J.C. Willey has equity interest and is consultant for Gene Express, Inc. E.L.Crawford has equity interest in Gene Express, Inc.

Design and validation of a robust diagnostic assay (prv-1 gene) based on real-time RT-PCR.

Häusler P., Bohle V.

Kooperationsgemeinschaft molekulare Labordiagnostik, Germany

Email: phausler@oncoscreen.com

Quantitative PCR of the prv-1 mRNA was set up and validated as a robust and reliable method for clinical routine diagnostics of Polycythemia vera rubra (PV) on the basis of peripheral blood specimens. Initially, the conditions of blood storage and shipping were evaluated. Then an optimised technique for cDNA preparation from granulocytes was established, followed by the identification of a panel of three reference genes out of a panel of six candidate reference genes, among them genes that had been suggested previously for hematologic diseases by the Europe Against Cancer group (EAC). This primary selection identified beta actin and G6PD as optimum reference genes that are clearly superior to B2M, GUS or ABL. The ranking of GAPDH remains controversial and depends on the mathematical model used for evaluation. On the basis of subsequent stability data where blood was stored at diverse ambient temperature the expression GAPDH proved to be rather unstable upon storage. On the basis of these data an assay was designed and validated. Limit of detection, limit of quantification, intra assay variability and inter assay variability were evaluated, respectively. Finally, a schedule for the collection of long term stability data was set up. This assay now needs to be clinically validated in a prospective trial. The modus operandi outlined here we suggest as a standard procedure for designing diagnostic assays based on quantitative real-time PCR.

Assessment of yeast intron insertion on expression and mRNA level of the human alpha-1 Antitrypsin cDNA in Pichia pastoris.

Hasannia S.1, Lotfi A. S.2, Mahboodi F.3 and Mohsenifar A.4

1The University of Guilan, Iran (Islamic Republic of), 2The Tarbiat Modares University, 3Pasture Institute of Iran and 4Medical Sciences of Semnan University

Email: s_hasannia@guilan.ac.ir

Human Alpha-1 antitrypsin (AAT) is a Serpin (serine Protease inhibitor) and an inhibitor of neutrophil elastase. This glycoprotein consists of 12% carbohydrate, 394 amino acids and has a molecular weight of 54 KD. As this protein is glycosylated and has a high MW. Any procedure which can increase the expression and secretion of the recombinant protein in yeast can be a useful in the production of this medicine. Although various methods, such as using various promoters, selection of specific untranslated region (UTRs), various signal sequences, appropriate inducer various mutants and other biotechnological procedures are being employed to increase recombinant protein expression in yeast. In this study a new technique which concentrates upon regulation in the nucleus and uses yeast intron integration in the human AAT cDNA, has been used to study its effect on gene expression. There are various report indicating a role for intron sequence in the expression of homologous and heterologous protein expression in animal and plant cells. Increased recombinant protein expression due to introns is termed intron-mediated enhancement or IME.

In this study, the yeast Pichia (strain GS-115) and the integrating shuttle vector pHIL-S1 has been used. At first, the pHIL-S1 vector carrying AAT was introduced into the yeast using the chemical – electroporation method and the resultant strain named GS-AAT. In order to integrate the yeast intron in a suitable site in the AAT cDNA, segment of exon II, yeast intron and multi-exon segments III, IV and V were separately amplified using specific primers for PCR and then these sequences were inserted into the bacterial vector pBluescript KS. Then the cDNA carrying the intron was separated from the vector and integrated in the pHOL-S1 vector and subsequently transferred to the yeast (strain GS-AATint). The two strains of yeast obtained were cultured in the same conditions and in media specific using SDS-PAGE and TIC, which is highly specific for assessment of inhibitory activity of AAT. In order to measure the cytoplasmic concentration of the related mRNAs in the two strains, Pfaffl method (relative quantitative RT-PCR) with SYBR green were used. Recombinant protein expression in both strains could be assessed after the second day and this indicates the yeast had carried out the splicing correctly. A comparison of the expression at 72 hrs in two media using the transformed yeast with TIC showed a 23 fold increase in the strain carrying the intron relative to the strain not carrying the intron. On the other hand, results obtained from RT-PCR show that the concentration of the cytoplasmic mRNA carrying the intron has increased 35 fold. The results obtained showed that integration of yeast intron in human cDNA in yeast can cause an increase in expression and subsequently secretion and can be used as a useful technique producing recombinant glycoproteins in Pichia pastoris.

High Sensitivity QuantiGene 2.0 Assay for Direct Quantification of mRNA Transcripts.

Son Bui, Nina Nguyen, Yunqing Ma, Quan Nguyen, George Zheng, Jessie Wu, Wen Yang, Botoul Maqsodi, Joan Davies, Jason Li, Gary McMaster, Frank Witney, Yuling Luo

Panomics, Inc., United States of America

Email: yluo@panomics.com

QuantiGene assay is a branched DNA signal amplification technology that quantitatively measures mRNA directly in cell lysates and tissue homogenates without the need for RNA isolation, reverse transcription, or target amplification. Although the assay has demonstrated the best precision and accuracy among all platforms evaluated in the MAQC (MicroArray Quality Control) study, the assay detection sensitivity is around 6,000 copies, limiting its application in low copy mRNA detection situations. We now report the development of QuantiGene 2.0. The assay maintains the same level of precision and accuracy as QuantiGene, but with increased detection sensitivity now below 200 copies for every gene tested. QuantiGene 2.0 has been used in quantifying mRNA in purified total RNA, cell lysates, and tissue homogenates. Furthermore, QuantiGene2.0 has proven to be well suited for mRNA quantification in FFPE (Formalin-Fixed and Paraffin Embedded) and whole blood samples with exceptionally high sensitivity. Side-by-side comparison of assay performance demonstrated that the assay sensitivity of QuantiGene 2.0 is now comparable to, and in many cases, better than qPCR.

Tracing the source of faecal pollution in water by qPCR: quantitative microbial source tracking (QMST).

Georg H. Reischer1, David C. Kasper1, Ralf Steinborn2, Robert L. Mach1, Andreas H. Farnleitner1

1Institute for Chemical Engineering, Gene Technology Group, Vienna University of Technology, Vienna, Austria and 2Institute of Animal Breeding and Genetics, Department for Animal Breeding and Reproduction, University of Veterinary Medicine, Vienna, Austria

Email: reischer@mail.zserv.tuwien.ac.at

One special field of application for qPCR that has only recently been developing is quantitative microbial source tracking (QMST) in water. QMST allows the allocation of faecal pollution in water to a specific source group e.g. human or animal sources by detection of source-specific faecal microorganisms. This information is vital for target-oriented measures for remediation of faecal influence on waters resources used for various purposes such as drinking water abstraction or recreation. The two most crucial requirements for such methods are the source-specificity (presence of the marker target in the faeces of one group, absence in all other faecal sources) and the sensitivity (high prevalence and abundance of the target organism in the specific source, sensitivity of the detection method). In order to meet those requirements we recently developed two qPCR methods for the source specific detection and quantification of two QMST markers specific for human (BacH) and ruminant (BacR) faecal input, respectively. The methods were developed for application in an alpine karst spring catchment area. The low levels of faecal contamination present in this environment demanded detection tools with a sensitivity way beyond the hitherto available methods. The assays detect faecal 16S rRNA gene markers from the bacterial phylum Bacteroidetes using TaqMan MGB probes. The qualitative and quantitative detection limits of the assays were 6 and 30 marker copies respectively. Source-specificity was found to be very high when applying the methods on hundreds of faecal DNA extracts from various sources from Eastern Austria. We determined marker equivalent concentrations in the range from 6.6 times 109 to 9.1 times 1010 BacH marker equivalents per g of human faeces and 0.9 to 3.9 times 109 BacR marker equivalents per g of ruminant faeces. The methods were sensitive enough to detect a few hundred pg of human faeces and a few ng of ruminant faeces in faecal suspensions after filtration and DNA extraction. To get an impression of the applicability on a larger geographically scale, the human-specific BacH qPCR assay was used to investigate samples taken along the whole length of the Danube River and from its important tributaries. The results obtained by the QMST BacH approach were the first to show high correlations with classical faecal indicator parameters in a QMST study. The ability to perform QMST studies offered by the newly developed methods will facilitate the task of identifying and locating sources of faecal pollution in water. After further evaluation in other regions it should be applicable on any geographical or temporal scale and allow quantitative estimation of the absolute contribution of the specific pollution sources. In addition it could provide necessary additional data for quantitative microbial risk assessment required to ensure safe use of water resources.

Preliminary evaluation of the GeneXpert Dx System for CML patients monitoring through the Xpert BCR-ABL Monitor 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.

Session:   High throughput quantitative PCR

Chair:   N. Zoric / L. Warren

Lecture hall   HS 15

Massively Parallel, Nanoliter-scale PCR for High Throughput Genomics.

Brenan C.

BioTrove Inc., United States of America

Email: cbrenan@biotrove.com

There is an increasing need for accurate and precise high throughput SNP genotyping and quantitative analysis of gene expression in diverse areas as pharmaceutical research and discovery, agricultural biotechnology, environmental testing and public health. In response, BioTrove has developed and is commercializing a nanofluidic platform uniquely capable of meeting these emerging requirements by enabling rapid, high throughput PCR assays in a nanoliter format. Called the OpenArrayTM, the device consists of a rectilinear array of 3072 through-holes in a microscope slide-sized stainless steel platen. Polymer coatings making the interior surface of each hydrophilic and the exterior surface of the platen hydrophobic are applied and allow PCR to be performed in each fluidically isolated 33 nL through-hole. After an overview of the OpenArray technology, a number of biomedical applications will be described based on SNP genotyping with the Taqman PCR assay and quantitative measurement of gene expression with real-time SYBR Green PCR.

Towards High-Throughput Single-Cell Expression Analysis.

Warren L.

Stanford University, United States of America

Email: luigiw@stanford.edu

Understanding the developmental program by which stem cells differentiate into diverse cell types is a central problem in current biology, the solution to which holds great therapeutic promise. Fluorescence-Activated Cell Sorting (FACS), a high-throughput single-cell gene expression assay, has for decades been the key enabling technology spurring progress towards this goal. By mapping cell population structure in gene expression space, FACS has made it possible to outline the branched sequence of fate decisions leading from the hematopoietic stem cell to the specialized effector cells of the blood. Because FACS measures surface-protein expression, it does not provide direct insight into the decision-making logic driving cellular differentiation, which is mediated by transcription factors acting in the nucleus. With today's technology, the status of this gene regulatory network is most readily characterized at the RNA rather than the protein level. Quantification of mRNA in individual cells remains challenging, especially if we seek to measure low-abundance messages (such as those for transcriptional regulators), assay multiple targets in the same sample, and collect data on enough cells to resolve distinct sub-populations. A new technique, microfluidic digital RT-PCR, facilitates quantitation of rare transcripts in single cells, and is compatible with low-order multiplexing using TaqMan chemistry. Two-stage qRT-PCR is another, less costly approach which relies on standard qPCR technology. In this method, a multiplexed RT-PCR with a limited number of amplification cycles is followed by independent simplex qPCR analyses on aliquots of ‘pre-amplified’ first-round product. The protocol is labor-intensive, however, limiting throughput. It will soon be feasible to combine two-stage qRT-PCR with microfluidic chip technology to support large-scale gene expression surveys, enabling multi-parametric, cross-sectional analysis of regulatory network state in progenitor cell populations. This should significantly benefit developmental studies.

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.

A GPCR brain map using Taqman Low Density Arrays.

Samaha R.

Applied Biosystems, United States of America

Email: samaharr@appliedbiosystems.com

The G Protein-Coupled Receptors (GPCRs) are a broad class of transmembrane proteins. A large percentage of successful small molecule drugs specifically target proteins in this class. Our study is designed to generate an expression map of 368 GPCR genes in the human brain. We used the Applied Biosystems Human GPCR Panel Taqman® Low Density Array (TLDA) and RNA samples from whole brain and 35 different brain sub-regions to generate this map. A total of 72 TLDA cards, 27,648 individual qRT-PCR reactions, were run in this study. To make it possible to run this large number of qRT-PCR assays from limited starting samples, we used a multiplex preamplification strategy (Taqman® PreAmp Master Mix & TLDA matched oligos). We present a performance analysis for the GPCR TLDA and preamplification methodology as well as the GPCR brain map. Several genes show interesting tissue distributions constituting potentially interesting drug targets.

BioMark™ System: A Breakthrough Real-time qPCR System for HT Expression Profiling, MicroRNA Analysis, and Single-Cell qPCR.

Unger M.

Fluidigm Corp., United States of America

Email: marc.unger@fluidigm.com

The BioMark System miniaturizes and highly parallelizes qPCR in nanofluidic circuits called dynamic arrays and digital arrays. Dynamic arrays enable parallel multiplexing of up to 48 genes for each of 48 samples yielding 2,304 ten nanoliter qPCR reactions per run. The dynamic array configuration is completely flexible allowing scientists to input any set of 48 samples and any set of 48 genes. The efficiencies realized in terms of time, effort, and cost are revolutionary compared to traditional microplate based systems. Digital arrays are a novel tool for easily and reliably performing digital PCR: single target molecule PCR by limiting dilutions. Digital arrays provide single-copy sensitivity combined with extreme accuracy to enable new applications such as single-cell gene expression analysis and somatic cell mutation detection.

Speed matters – Fast ways from template to result.

Thorsten Traeger

QIAGEN GmbH, Hilden, Germany

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. This talk will focus on how to overcome the challenges of achieving fast PCR, both in real-time PCR and RT-PCR analyses, using a novel technology that is fully compatible with existing PCR assays and allows fast PCR on standard as well as fast PCR platforms. Furthermore, an alternative approach to speed up the experimental workflow when analyzing cultured cells will be presented. This approach eliminates the need for RNA purification and allows real-time RT-PCR analysis direct from cell lysates.

The presentation will expound the principles of the technologies and will show data demonstrating successful real-time and end-point PCR analyses with time savings of up to 70%. The real-time PCR data will include data generated using different detection formats, such as SYBR Green dye and sequence-specific probes in single and multiplex PCR.

Identification of the vascular lineage-specific transcriptome and development of a novel low-density microvascular differentiation array.

Jay W. Shin1, Kentaro Kajiya1, Weiniu Gan2, Peter Li2, Rainer Kunstfeld3 and Michael Detmar1

1Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland, 2Molecular Biology Division, Applied Biosystems, Foster City, CA, USA and 3Department of Dermatology, Medical University Vienna, Vienna, Austria

Email: jay.shin@pharma.ethz.ch

Angiogenesis and lymphangiogenesis have important roles in cancer progression and inflammatory diseases, but it has been a challenge to evaluate these processes quantitatively. Using two different microarray platforms, we first identified the comprehensive lineage-specific transcriptomes of human lymphatic (LEC) and blood vascular (BVEC) endothelial cells. We identified 236 lymphatic signature genes and 342 signature genes for blood vascular endothelium. In silico analyses of the biologic pathways associated with these genes revealed lineage-specific functions for each cell type. Using a selection of 85 identified lineage-specific genes, we developed a TaqMan RT-PCR-based, microfluidic card-formatted low-density microvascular differentiation array (LD-MDA) that can be used to reliably identify and quantify endothelial cells based on their lineage-specific differentiation. Importantly, using LD-MDA, we were able to quantify levels of angiogenesis and lymphangiogenesis in tissue samples from patients with chronic inflammatory skin disease, indicating that this assay might be developed as a novel tool for the rapid and quantitative evaluation of pathological (lymph)angiogenesis in clinical and pre-clinical studies.

GeXP - a new approach in high-throughput gene expression analysis.

Han-Chang Chi1, Yong Wu1, Jane Luo1, Kahuku Oades2, Gordon Vansant2, Scott K. Boyer1, Manfred Souquet1 and Keith Roby1, 1Nucleic Acid Testing Business Group, Beckman Coulter, Inc., 4300 N Harbor Blvd, Fullerton, CA 92834 and 2Analytical Services, Althea Tech

Beckman Coulter, Germany

Email: msouquet@beckman.com

Quantitative gene expression analysis is playing an increasingly important role in cancer research. Currently available techniques either utilize microarray to detect the expression of a high number of genes per reaction at high cost, or utilize real-time PCR† to detect the expression of a few genes at low throughput. A multiplex approach to analyze a set of multiple genes from a given biological pathway in a single reaction using a limited amount of total tissue RNA is of great interest to cancer biologists. To address this need, the GenomeLab™ GeXP Genetic Analysis System was developed utilizing eXpress Profiling, a patented, highly multiplexed PCR approach to quickly and efficiently look at the expression of 20-30 multiplexed gene sets with greater sensitivity. The throughput is scalable from 80 to over 4000 gene expression results per day. Here we present a study of two cancer panels: a set of 29 genes directly or indirectly related to breast cancer formation; and the second set of 20 genes for metastasis progression. These two multiplex gene expression marker panels were developed not only for breast cancer detection, but also to differentiate cancerous stages. Our study demonstrated that this novel approach can quantitatively detect the expression of both cancer marker panels in a single reaction using as little as 25 ng of total RNA isolated from human breast cancer tissues and other types of cancerous tissues. The GenomeLab GeXP System has proven to be a cost-effective way of performing multiplex gene expression analysis with scalable throughput capacity, high assay robustness, and excellent data quality.

† The PCR process is covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffman-LaRoche, Ltd.

Novel qPCR methods for cellular high throughput compound screenings of SOST expression inhibitors.

Angela Furrer, Simone Degen, Heidi Jeker, Johann Wirsching and Hansjörg Keller

Bone & Cartilage Unit, Musculoskeletal Disease Area, Novartis Institutes for BioMedical Research, Basel, Switzerland

Email: hansjoerg.keller@novartis.com

The SOST gene encodes for sclerostin, an osteocyte-secreted important negative regulator of bone mass. Lack of SOST expression in bone causes the rare human genetic disorders Sclerosteosis and Van Buchem disease, which are characterized by massive life-long bone overgrowth of the whole skeleton. Thus, inhibiting sclerostin expression represents a new opportunity for the development of strongly needed novel bone anabolic osteoporosis therapies. To this end, we have established a cellular assay for the compound screening of SOST expression inhibitors using SOST-expressing UMR-106 rat osteosarcoma cells and quantitative real-time RT-PCR (qPCR) analysis of SOST mRNA expression. Forskolin inhibited SOST expression within 6h by about 90% with an IC50 of 515 ± 121 nM. QPCR analysis was performed using purified RNA and two-step cDNA synthesis/real-time PCR, and, thus, did not permit high throughput application. Here, we report the testing of two novel methods for faster and simpler qPCR: The FastLane Cell Multiplex kit from Qiagen allowing one-step RT-PCR directly from cell lysates and another kit from Qiagen permitting fast RT-PCR using thermal cyclers with standard or rapid ramping rates. Method performances were investigated by determining SOST inhibition dose-response curves for forskolin and solvent control. Compared to the two-step qPCR assay with purified RNA, the one-step qPCR assay with cell lysates showed very similar data reproducibility and accuracy, but was much less laborious requiring less than half the amount of time. Solvent control data variation and forskolin IC50 values were not significantly different between the two methods. Cell lysates were stable at -80º C for at least one month allowing qPCR re-analyses or expression analyses of new genes. A test kit version for fast RT-PCR was also successfully tested yielding similar data reproducibility and variation. Solvent control data variation and forskolin IC50 values were not significantly different from the previous two methods. RT-PCR time requirement was further reduced with this method due the reduced cycling times, but this method still required RNA purification. In conclusion, novel qPCR methods were established that allow gene expression analysis using cell lysates without prior RNA isolation and that enable fast-cycling RT-PCR using normal thermal cyclers, respectively. These methods decisively accelerate and simplify cellular qPCR assays permitting facilitated automation and thus, they are well suited for use in high throughput compound screenings of gene expression regulators.

A comprehensive and quantitative way : HiCEP.

Abe M.

National Institute of Radiological Sciences, Japan

Email: abemasum@nirs.go.jp

qPCR is an ideal way to observe the expression of each transcript but is not suitable for comprehensive study, because of the limitation on its throughput. We have attempted to develop a new way to satisfy both requirements, namely highly quantitative and comprehensive. A PCR-based method called AFLP was developed for analyzing numerous DNA fragments formed by restriction-enzyme digestion. Its core technology is a unique PCR step for dividing the whole DNA-fragments population into subpopulations through selective amplification. It is called as ‘selective PCR’ but it had worked still partially. We found that there is temperature point, at which ‘selective PCR’ works nearly perfect. Around 71.5 ºC is good for the annealing temperature where only few miss annealing occur. Cloning and sequencing of more than 15,000 peaks detected in mouse embryonic stem (ES) cells revealed that 96.2% of them were generated with correct selection. In combination with using restriction enzymes, this nearly-100% accurate selective PCR enables us to distinguish huge DNA fragments without any sequence information. This procedure is applicable to transcriptome analysis, genome analysis such as restriction-enzyme mapping and comprehensive DNA methylation assay and so on. Using this procedure, more than 40,000 transcripts including about 2,000 of novel transcripts, were identified in mouse ES cells and the expression of each transcript was measured quantitatively. It is notable that this new procedure can identify unknown transcripts as well as known ones. Non-coding transcripts are often detected. Currently, 100 cells are enough for the analysis.


Session:   qPCR NOS Session

Normalization & Optimization & Standardization

Chair:        T. Nolan / P. Pinzani

Lecture hall   HS 15

Quantification of mRNA using the real-time RT-PCR.

Tania Nolan1, Rebecca E Hands2, Stephen A Bustin2

1Sigma Aldrich, United Kingdom and 2Queen Mary University of London, London, UK

Email: tnolan1@europe.sial.com

The real-time reverse transcription (RT) polymerase chain reaction (PCR) (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology, diagnostics and other areas and has become the method of choice for the quantification of mRNA. Although often described as a “gold” standard, it is far from being a routine assay.

Current problems

The widespread use of this technology has resulted in the development of numerous protocols that generate quantitative data using

• fresh, frozen or archival (formalin-fixed, paraffin-embedded) samples

• whole tissue biopsies, microdissected samples, single cells, tissue culture cells

• total or mRNA

• a range of different cDNA priming strategies

• different enzymes or enzyme combinations

• assays of variable efficiency, sensitivity and robustness

• diverse detection chemistries, reaction conditions, thermal cyclers and

• individual analysis and reporting methods.

This obvious lack of standardisation at every step of the assay is exacerbated by significant differences in sample processing, use of controls, normalisation methods and quality control management and has serious implications for the reliability of the assay, relevance and reproducibility.

The significant problems caused by variability of RNA template, assay design and protocols as well as inappropriate data normalisation and inconsistent data analysis are widely known, but also widely disregarded. As a first step towards standardisation, a comprehensive RT-qPCR protocol is described that illustrates the essential technical steps required to generate quantitative data that are reliable and reproducible.

qPCR pitfalls - primer and probe design / fluorophore quencher combinations.

Beslin C.

Eurogentec, Belgium

Email: cl.beslin@eurogentec.com

When designing a qPCR assay, different parameters have to be taken into account such as samples/experimental group, type of controls, design and synthesis of the primer and probe, method of RNA extraction (quality and purity of RNA), reverse transcription (one step or two step) and qPCR (singleplex or multiplex). All these steps are commonly discussed except the design of probes and primers.

Primer and probe design is a crucial step in your experimental design; time spent in the design of the primers and probes will save time in the optimisation of the assay. Design, not only includes the basic rules, pitfalls or tricks to design primers and probes but also a combination of the right fluorophores and quenchers: which fluorophore should be chosen, which quencher should be combined, how to multiplex them. These choices are not just dependant on your assay and your instrument but also on the available synthesis techniques. Design pitfalls and tricks for various instruments and fluorophore-quencher combinations will be discussed.

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. Using this assay as an example, we provide a step-by-step tutorial on the considerations that go into the design and optimization of multiplexed PCR: selecting sequences, optimizing reactions, and characterizing amplification performance. By sensitively exposing the presence of these organisms, we highlight the capability of multiplexed qPCR.

Accurate quantification of mRNAs and housekeeping gene selection for quantitative real-time RT-PCR normalization in European beech (Fagus sylvatica L.) during abiotic and biotic stress.

Olbrich M.

GSF, Germany

Email: maren.olbrich@gsf.de

Analyses of different plant stressors are often based on gene expression studies. Quantitative Real-time RT-PCR (qRT-PCR) is the most sensitive method for the detection of low abundance genes. However, a critical point to note is the selection of housekeeping genes as an internal control. Many so-called ‘housekeeping genes’ are often affected by different stress factors and may not be suitable for use as an internal reference. We tested seven housekeeping genes of European beech by qRT-PCR using the Sybr Green PCR kit. Specific primers were designed for 18S rRNA, actin, GAPDH1, GAPDH2, α-tubulin, and ubiquitin-like protein. Beech saplings were treated with increased concentrations of either ozone or CO2. In parallel, the expression of these genes was analyzed upon pathogen infection with Phytophthora citricola. To test the applicability of these genes as internal controls under realistic outdoor conditions, sun and shadow leaves of 60-year-old trees used as a comparison. The regulation of all genes was tested using a linear mixed-effect model of the R-system. Results from independent experiments showed that actin was the only gene not affected by any treatment tested. The expression of the other housekeeping genes varied more or less with the degree of stress applied. These results highlight the importance of undergoing an individual selection of internal control genes for different experimental conditions.

EvaGreen: a new fluorescent nucleic acid dye for real-time qPCR.

Mao F1,2, Leung WY1, and Xin X.1,2

1Biotium, Inc., Hayward, US and 2AlleLogic Biosciences Corporation, Hayward, US

Email: shanex@allelogic.com

EvaGreen (EG) is a newly developed DNA-binding dye that is particular useful for both quantitative real-time PCR (qPCR) and post PCR DNA melt curve analysis. Compared with SYBR Green I (SG), EG was found to have less PCR inhibition and thus could be used at a relatively high concentration (0.67-1.33 uM), which enabled PCR to yield more robust signals without adversely affecting the specificity and amplification efficiency. The use of relatively high EG concentration also eliminated so-called dye redistribution problem, making the dye suitable for use in high-resolution melt curve analysis. Furthermore, the relatively low PCR inhibition of EG also permitted fast cycling protocol in qPCR without sacrificing PCR specificity and reproducibility. Because EG has excitation and emission spectra similar to those of SG and FAM, the dye is compatible with nearly all commercial qPCR instruments. Finally, EG was shown to be nonmutagenic, cell membrane-impermeable and extremely stable, thus facilitating its use and handling.

Quantitative, multiplexed amplification with the Plexor™ qPCR Systems.

Katharine Hoffmann, Benjamin Krenke, Cynthia Sprecher, Susan Frackman, Ethan Strauss, and Douglas Storts

Promega Corporation, United States of America

Email: doug.storts@promega.com

The Plexor™ qPCR, One-Step qRT-PCR, and Two-Step qRT-PCR Systems enable quantitative, multiplex amplification experiments on a variety of real-time instruments. The system consists of free web-accessible primer design software, reagent systems and free data analysis software. The Plexor™ Systems work by measuring a reduction in fluorescent signal during amplification. Amplification uses only two primers, one of which contains a modified base with a fluorescent reporter on the 5'-end. As amplification proceeds, fluorescence is reduced by site-specific incorporation of a fluorescent quencher (attached to a modified nucleotide) inserted opposite the complementary modified base. The quencher is in close proximity to the fluorescent dye located on the 5'-end of the primer, resulting in a reduction in the fluorescent signal. After PCR, a melt analysis can be performed to provide an internal control for the final assay design or to expedite troubleshooting during assay development. We will present data demonstrating reliable multiplexed amplification results, over a broad range of target concentrations.

Taqman vs Molecuarl Beacon. Design and optimisation of an improved probe for real-time PCR detection.

R. Powell1, T.Brown2, J. Wicks1

1PrimerDesign Ltd, United Kingdom and 2Southampton University, Organic Chemistry

Email: rob@primerdesign.co.uk

A novel probe (PerfectProbeTm) has been designed which incorporates the best functional characteristics of Taqman and molecular beacons (MB) probes. These probes have a “shared stem” hair pin structure and are therefore optimally quenched like MB probes. However they are cleaved, rather than displace, during amplification like the Taqman probe. These characteristics combine to produce a probe that is more sensitive at detecting amplification than either Taqman or Molecular Beacon probes. The novel probe produces earlier cycle threshold values when compared directly to the reporting of Taqman or MB probes. They also give rise to much higher signal to noise ratios and are more flurogenic. The critical design criteria for these probes will be presented and a model for understanding their functioning during real-time PCR amplification.

Comparison of different probe chemistries and platforms to improve the sensitivity of real-time PCR.

Reynisson E.1,2, Josefsen M.H.3, Krause M.3, Hoorfar J.3

1Matís, Iceland, 2University of Iceland and 3Danish Institute for Food and Veterinary Research (DFVF)

Email: eyjolfur@matis.is

A validated qPCR-based Salmonella method targeting a 94-bp sequence of the ttr gene was used as a model to compare six different combinations of reporter and quencher dyes of a TaqMan probe, on three different instruments, to improve the detection limit in a realtime PCR assay with the aim of a same-day analysis. The use of locked nucleic acids (LNA) and Scorpion probes were also tested. The combination FAM–BHQ1 or Cy5–BHQ3, both dark quenchers, gave the best results (Cycle threshold (Ct) of 25.42 ± 0.65 and 24.47 ± 0.18 at 103 DNA copies). When comparing different probe technologies, the LNA probe (FAM–BHQ1) was the most sensitive with the strongest fluorescence signal (dR last 48066), resulting in 0.6 to 1.1 lower Ct values than TaqMan probe, and 1.9 to 4.0 lower Ct than the Scorpion system (FAM–BHQ1). The RotorGene real-time PCR instrument gave 0.4–1.0 lower Ct values (more sensitive) than the Mx3005p, and 1.5–3.0 lower than the ABI 7700. Using the LNA in a RotorGene instrument, we detected the following Salmonella DNA copies in 1-ml pre-enriched samples: fishmeal (100 copies), chicken rinse (100 copies) and pig feces (10 copies). The detection probability of the final assay on inoculated fecal samples was 100% at 2 x 104 copies per ml. In conclusion, the LNA probe with annealing temperature of 65 °C could be useful for more sensitive detection limits.

Sample number and denaturation time are crucial for the accuracy of capillary-based LightCyclers.

Thomas von Kanel, Florentin Adolf, Mircea Schneider, Javier Sanz, Sabina Gallati

Division of Human Genetics, University of Berne, Switzerland

Email: biotom@students.unibe.ch

Background: While performing real-time PCR for deletion and duplication screenings on a capillary-based LightCycler instrument, carousel-position variations in cycles over threshold Ct were observed. These variations can reach an extent indicating a duplication or even a deletion in a diagnostic setup (maximum Ct difference between different carousel positions > 0.5).

Methods: Protocol amendments such as complete filling of the carousel with capillaries and increased denaturation time during PCR cycling were tested for their potential to increase the accuracy of LightCycler instruments.

Results: Highest accuracy of the LightCycler instruments was obtained by complete filling of the carousel and by increasing the denaturation time to 5 seconds during cycling, with coefficients of variation dropping from ≥ 0.68% (unfilled carousel, 0 seconds denaturation) to ≤ 0.23% (filled carousel, 5 seconds denaturation), thus minimizing the chance to obtain false-positive results due to positional effects.

<>Conclusions: In order to achieve the precision necessary to perform reliable deletion and duplication screenings and copy number variation detection, we highly recommend complete filling of the LightCycler carousel with capillaries, independent of the number of reactions performed, and to set the denaturation time to 5 seconds. In addition, we advise to check any real-time PCR assay for eventual instrument position dependent effects – not only on capillary-based cyclers, but also on plate-based platforms.

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