Postmortem mRNA Profiling: Possibilities and Limitations

Marielle Heinrich, Antje Koppelkamm
University of Freiburg, Germany

Abstract
Molecular investigations gain increasing interest in forensic medicine. Examination of gene expression levels at the time point of death might shed further light into the cause and circumstances of death. This includes pathophysiological conditions of disease and injury as well as the duration of agony. Additionally, recent developments in forensic genetics revealed that tissue specific mRNAs can be used to determine the type of body fluid present in a crime scene stain. Although RNA is known to be rather instable, we were able to extract RNA in adequate quality from tissue samples collected during medico-legal autopsy. Nevertheless, working with human post-mortem tissue means to deal with highly variable RNA integrities. The aim of the present study was to identify parameters that have to be considered during quantitative gene expression analysis. Therefore, we analysed the RNA qualities of human autopsy samples and investigated the influence of RNA integrity on RT-qPCR results. Additionally, we aimed to identify pre- and post mortem parameters, which might influence the RNA integrity. Consequences for the informative value of quantitative gene expression data will be discussed.


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Zip Nucleic Acid hydrolysis and hybridisation probes for improving detection

Nathalie Lenne, Clément Paris, Valérie Moreau, Gaëlle Deglane, Patrick Erbacher
Polyplus-transfection, France

Abstract
Zip Nucleic Acids (ZNAs) are oligonucleotides with attached cationic units that reduce electrostatic repulsion between nucleic acid strands. The modification improves hybridisation by accelerating the target recognition and increases the melting temperature of the oligonucleotide without altering the specificity. The presentation will describe the performance of ZNA dual-labelled qPCR probes, which are alternately hydrolysis or hybridisation probes depending on the localisation of the cationic units. ZNA hybridisation probes enable SNP genotyping by probe melting analysis.


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New technologies for FFPE samples: Improved RNA isolation and novel cDNA priming for RT-qPCR and for microarray analysis

Guido Krupp 1, Susanne Quabius 2, Rolf Jaggi 3
1 AmpTec GmbH, Hamburg, Germany; 2 Institute Immunology, UKSH Kiel, Germany; 3 Department of Clinical Research, University of Bern, Switzerland

Abstract
Archival FFPE samples harbour a wealth of information. Although FFPE RNA is severely degraded plus challenges due to inter- and intramolecular cross-linking and base modifications, mining of gene expression data is still possible and extracted information about differential gene expression is comparable to data from frozen samples, even at quantitative level. The combination of a novel procedure for RNA liberation and demodification results in highly reproducible data in RT-qPCR studies (Oberli et al 2008) and can be used to determine RNA profiles of cancer samples (Schobesberger et al 2008). Our novel TR priming combines the advantages of oligo-dT (priming near the 3’end and selection against rRNAs) with random priming (works with mRNA fragments without poly-A: no requirement for a universal target sequence). Oligo-dT primers provide cDNA from regions next to the 3‘-poly-A tail. Internal transcript regions are (partially) lost and effects on different mRNAs and PCR amplicons are variable. Accordingly, gene expression results (RT-qPCR) vary widely with different RNA qualities. For example, upon RNA degradation, the Ct for actin mRNA increases 9.9 cycles, with much lower effects on other mRNAs, e.g. an increase of only 3.9 for YMHAZ. Although there is good selectivity against rRNA sequences in intact RNA: Ct for 18S rRNA is only -4.1 vs actin mRNA (17-fold more rRNA). Since rRNA is more stable, the difference increases to 9.0 in degraded RNA (500-fold more rRNA). With random primers (or a mix of random and oligo-dT), the recovery of internal transcript regions is improved. Accordingly, there is less variability with different RNA qualities: Ct for beta-actin increases by 5.4 cycles, comparable to 4.7 for YMHAZ. Selectivity against rRNA sequences is poor. Intact RNA: Ct for 18S rRNA vs actin is -9.2 (588-fold more rRNA), with further increase to -10.7 (1663-fold more rRNA). With our novel TR primers, preferential priming occurs at the 3‘-end of RNA fragments (independent from poly-A) and different to random priming, further “cutting“ of RNA fragments into multiple, short cDNAs is prevented; combined with improved recovery of internal transcript regions. Ct for beta-actin increases by 4.1, very comparable to 4.4 for YMHAZ. Furthermore, selectivity against rRNA sequences is good. Intact RNA: Ct for 18S rRNA vs actin is -7.5 (181-fold more rRNA), a small increase to -8.6 (388-fold more rRNA, lower than for oligo-dT with this RNA sample). TR priming can be used for FFPE RNA templates, followed by qPCR analyses or combined with mRNA amplification and labelling for genome-wide microarray gene expression profiling. Selectivity against rRNA sequences makes TR primers an ideal tool for the novel “Exon Microarrays“. Presented data will demonstrate advanced recovery of FFPE RNAs: RNA profiles (Agilent Bioanalyzer), comparison of expression profiles obtained with RNAs from fresh-frozen and from FFPE samples, as determined by RT-qPCR and by Affymetrix microarrays.


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Advanced qPCR – one step ahead with next generation dyes

Alexander Rácz
Eurofins MWG Operon, Germany

Abstract
Over the past 20 years, quantitative polymerase chain reaction (qPCR) has become a powerful tool for nucleic acid quantification. Together with the development of a broad range of applications for gene expression or mutation analysis, there was a continuous evolution of instrument hardware, detection formats or strategies and consumables. Hybridisation probes like DualLabeled Probes (DLP´s), Molecular Beacons or FRET-Probes have several advantages over unspecific intercalation dyes. In this talk we describe the impact of dyes and quencher molecules on the performance of qPCR experiments and present the features and benefits of next generation dyes for qPCR.


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Hydrolysis probes with improved quenching and performance

Mark Aaron Behlke
Integrated DNA Technologies, Inc., United States of America

Abstract
Fluorescence-quenched probes are used in a wide variety of molecular biology applications today and have particular utility in qPCR. Efficient quenching is crucial for optimal probe performance. A new dark quencher (ZEN) has been developed which can be placed internally within hydrolysis probes, permitting the quencher to be positioned very close to the fluorophore, thereby improving quenching and probe performance. Most chemical groups are destabilizing to duplex formation when introduced internally within a sequence; the new ZEN quencher stabilizes duplex formation and thus increases Tm of the probe instead of lowering Tm as would normally be expected.


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Dye spectra, dynamics and consequences

Roderic Fuerst
IT-IS Life Science, Ireland

Abstract
The process of qPCR requires robust and reliable florescence monitoring of multiple dyes. Commonly qPCR systems use discrete excitation and emission filters in the monitoring of amplification and hybridization florescence. Also, algorithms used for resolving components of multiplex assays rely on the assumption that dye spectra remain constant during PCR and subsequent melting point analysis. In some cases this assumption is not valid. The nature and consequences of different approaches will be discussed.


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A Practical Approach to Assay Design for qPCR – Overcoming Difficult Assays, Designs and Optimizations while Conforming to the MIQE Guidelines

Francisco Bizouarn
Bio-Rad Laboratories, United States of America

Abstract
Quantitative PCR assays in the past have often been run without serious specificity and quality considerations. Target sequence information was inserted into “black-box” software packages and assays run under general conditions. With the arrival of MIQE guidelines, a greater emphasis is placed on the use of robust and validated assays. Generating robust assays is generally a simple task, but occasionally obstacles can be encountered. Ease in the optimization of an assay is often directly linked to the original design. In this talk, we will present critical assay design parameters and tips to overcome potential problems, as well as quick optimization and validation strategies that help fulfil MIQE guidelines.

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Why the need for qPCR publication guidelines? – The case for MIQE

Stephen Andrew Bustin
Barts and the London School of Medicine and Dentistry, United Kingdom

Abstract

MIQE (the minimum information for the publication of quantitative real-time PCR experiments) was conceived as a set of guidelines to encourage more comprehensive communication of experimental detail within publications utilising qPCR technology. Although MIQE is beginning to have an impact on the quality of data published, some journals, frequently with high impact factors, continue to publish misleading and technically inadequate research papers. MIQE is not a pedantic set of instructions that makes publication a more arduous chore. Instead, MIQE’s extensive reference to and incorporation of the many variables that make up the pre-assay, assay and data analysis steps makes it an ideal template for novice and experienced user alike on which to design every future qPCR assay. A concerted effort to implement these guidelines is essential if qPCR is to mature into a robust, accurate and reliable quantification technology.

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