MONDAY, APRIL 21
8:00-8:45 am Conference Registration and Morning Coffee
8:45 Chairperson’s Opening Remarks
8:55 Quality Control, Discovery, Diagnostics and Target Validation: Exploiting qPCR Technologies Throughout the Laboratory
Andrew I. Brooks, Ph.D., Associate Professor, Environmental Medicine & Genetics, UMDNJ/Rutgers University
Quantitative PCR applications have been developed and proven to be useful in an ever expanding number of applications. Although quantitative PCR technology was utilized primarily for gene expression applications, the field has developed in many exciting new areas. Today quantitative PCR technology is being exploited for a variety of laboratory applications in both basic science and clinical settings; from sample quality control to molecular diagnosis. The focus of this presentation is to identify and describe the relevant applications for qPCR in a laboratory setting. We will focus on both basic and clinical applications and discuss the broad range of applications for which the technology can be employed. Specific qPCR protocols and workflows will be presented to help develop a comprehensive review of the importance of qPCR in the laboratory environment.
9:25 Putting it all Together: Assay Validation, Primer Selection, Reverse Transcription and mRNA Integrity
Stephen A. Bustin, Ph.D., Professor, Centre for Academic Surgery, Institute of Cell and Molecular Science, Queen Mary’s School of Medicine and Dentistry, University of London
Preassay validation is a critical section of any experiment designed to quantitate cellular mRNA. Choice and localization of RT primer, optimization of PCR primers, choice of RT and assessment of mRNA quality are all essential if mRNA quantification is to have any real relevance. This presentation will provide a detailed analysis of the priming and reverse transcription steps and demonstrate the difficulties associated with measuring of RNA integrity.
9:55 Quantitative Validation of Gene Expression Data: What are you Validating and Why
Steven Enkemann, Ph.D, Director, Microarray Core Laboratory, H. Lee Moffitt Cancer Center and Research Institute
Gene expression is a complex coordination of a number of cellular events. The mRNA concentration of any cellular transcript is controlled by the rates of transcription, processing, translation, and degradation, in addition to cellular location. Knowing how much mRNA is present is only the first step in understanding gene regulation. A microarray can provide more information than simply the amount of mRNA in a cell. Shouldn’t quantitative PCR also tell you more?
10:25 Networking Coffee Break
10:45 High-Throughput Primer Design and in Silico Assay Evaluation
Jo Vandesompele, Ph.D., Center for Medical Genetics Ghent (CMGG), Ghent University Hospital
Real-time PCR is the gold standard method for accurate and sensitive nucleic acid quantification. As this technology is frequently being used in high-throughput experiments typically analyzing a few hundreds of target genes, primer design is often a bottleneck. We recently integrated Primer3 with the proven RTprimerDB in silico assay evaluation pipeline, to enable researchers to design and validate real-time PCR assays in an automated manner using a simple web application. Results of a high-throughput real-time PCR experiment quantifying over 400 transcribed ultra-conserved regions will be discussed.
11:15 Comparison of Different Priming Strategies for cDNA Synthesis by Reverse Transcriptase as Measured by Real-Time RT-qPCR
Kevin L. Knudtson, Ph.D., DNA Facility, University of Iowa
The Nucleic Acids Research Group of the Association of Biomolecular Resource Facilities (ABRF) conducted a study to evaluate priming strategies for generating cDNA for use with real-time RT-qPCR. The goal of this study was to determine which single or combinatorial priming strategies were best suited for the most comprehensive synthesis of cDNA from total RNA for use in real-time RT-qPCR. The study evaluated the ability of random primers of increasing length (6 to 21 bases in steps of 3 bases), oligo-dT, anchored oligo-dT, combinations of random primer and oligo-dT, assay-specific primer and no primer with and without reverse transcriptase to initiate cDNA synthesis. Quantitative PCR was used to measure the relative levels of cDNA production by each primer combination. The results of this study will be presented.
11:45 Seeing is Believing? Development of a Rigorous Control Panel for qRT-PCR
Emi Arikawa, Ph.D., Scientist, R&D, SuperArray Bioscience Corporation
The exceptionally sensitive nature of quantitative reverse transcription-PCR (qRT-PCR) requires a set of rigorous controls to ensure the accurate interpretation of results. Many users unknowingly omit crucial validating steps. In addition, even with some controls, many experimental data are still beyond interpretation due to the incompleteness of those controls. SuperArray has developed a proprietary technology to ensure the quality of input RNA samples, to monitor the performance of reverse transcription and PCR, and to account for variations in PCR efficiencies in qRT-PCR analyses. We will describe a panel of control assays that can be easily implemented into any qRT-PCR experiment to provide quality-control checkpoints at each step. In addition, we will discuss some of the approaches currently employed to eliminate errors and biases resulting from incorrect assumptions in the analysis of real-time PCR data.
12:15 Luncheon Technology Workshop
The Use of Gene Expression in a Community Based Cohort
Kahraman Tanriverdi, PhD, Instructor of Medicine, Boston University School of Medicine, Whitaker Cardiovascular Institute
Recent data suggest that patients with both acute and chronic cardiovascular disease (CVD) have a heightened prothrombotic state and increased interactions between platelets and leukocytes. Platelet-leukocyte interactions lead not only to acute thrombotic occlusion, but also contribute to the chronic process of atherosclerosis. Additionally, recent studies suggest that platelets participate actively in inflammatory processes independent of their interactions with leukocytes. Our preliminary work evaluating platelets and peripheral blood mononuclear (PBMC) cells from patients with CVD, demonstrated receptors and enzymes stimulate the pro-inflammatory nuclear factor B (NF B) signaling pathway that enhances transcription of specific pro-inflammatory and thrombotic genes. We have collected 2300 blood samples from participants of offspring of Examination 8 from Framingham Heart Study. We have isolated platelets and PBMCs from citrated blood samples and performed RNA isolations. We have tested relative expression levels of 48 genes in platelet and PBMCs by using Dynamic Array 48.48 which is a high throughput Real-Time PCR system by Fluidigm, San Francisco, CA. We have determined the expression level of 48 genes for 46 samples in a few hours and we have performed 13,824 PCR reactions a day.
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2:00 Chairperson’s Remarks
2:05 Quantitation and Results Validity Analysis
John M. Clemens, Ph.D., Associate Research Fellow, Abbott Molecular
Implementation of real-time PCR in the clinical laboratory drives the need for automation of both processing and analysis steps. Conventional analysis of real-time PCR by Ct method is susceptible to periodic anomalies in the fluorescence response and often requires subjective analysis by a skilled reviewer. We have developed and automated a methodology for PCR analysis suitable for the IVD market that utilizes PCR curve shape analysis. The “maxRatio” method provides an independent measure of cycle number from traditional Ct analysis that allows it to be used as a validation of the Ct analysis or in place of it. It also provides a relative measure of reaction efficiency that is used for discrimination of reactive and non-reactive PCR populations and results validity evaluation. The talk will address an overview of the method, illustrate performance characteristics including challenges from typical signal anomalies and provide examples of clinical application.
2:35 Toward Ultimate Quantification - Recent Advances in Statistical Analysis of Real-time PCR Data with Linear Models
Joshua Yuan, Ph.D., Genomics Scientist, UTIA Genomics Hub, University of Tennessee
Linear models such as ANCOVA and multiple regression were first developed to derive deltadeltaCt, confidence level estimation, and p value estimation for the analysis of high quality real-time PCR data with amplification efficiency close to 100%. For low quality real-time PCR data, the amplification efficiency can be first estimated by the simple linear regression and then integrated into the unbalanced linear combination of Ct in the multiple regression model to estimate the efficiency-adjusted deltadeltaCt, the confidence intervals, and the p value for differential expression. Besides the gene quantification studies, simple linear regression and multiple regression models were also developed for the estimation of transgene copy numbers by real-time PCR, and the models can also be readily adapted for the absolute gene expression quantification and the applications such as viral infection load analysis.
3:05 FDA/ICH Analytical Method Validation for Genomic Transcript Abundance by Standardized Reverse Transcriptase PCR using a Standard Mixture of Internal Standards
John Anders, Ph.D., V.P. Operations, Operations, Gene Express Inc.
This report contains analytical method validation data for a multi-gene malignancy lung cancer interactive transcript abundance index (E2F1 x MYC / CDKN1A) obtained and analyzed by StaRT-PCR™ using SMIS™. This method validation was conducted under US FDA and International Conference on Harmonization (ICH) as described in the “Guidance for Industry; Q2B Validation of Analytical Procedures: Methodology, 1996. Transcript abundance measurements were acquired from cDNA following reverse transcription of total RNA. Analysis follows end point PCR amplification of calibrated cDNA using a single set of forward and reverse primers that co-amplify both native (NT) and internal standard (IS) transcripts in every transcript measurement. Detection of amplicon pairs (NT and IS) is achieved by rapid micro-electrophoretic separation with laser induced fluorescence. Validation data include, specificity, accuracy by recovery from spiked samples, intra-, inter-, day to day and lab to lab precision, linearity, sensitivity, range, robustness, system suitability and reagent stability for all genes of interest. The validation experimental procedures will be discussed with detailed data analysis all in an effort to support submission of a prognostic test for subsequent regulatory approval.
3:35 Technology Spotlight (Sponsorship Available)
3:50 Networking Refreshment Break, Poster and Exhibit Viewing
4:30 Core Managers Panel Discussion
High-throughput genomic data from gene expression experiments can be very complex. Listen and learn as Core Managers share their tips and techniques to overcome qPCR bottlenecks and provide useful demonstrated solutions to enable scientists to better evaluate their results.
Panelists:
Andrew Brooks, Ph.D., Rutgers University
Steven Enkemann, Ph.D., H. Lee Moffitt Cancer Center and Research Institute
Kevin L. Knudtson, Ph.D., University of Iowa
Christian Leutenegger, D.V.M., Ph.D., IDEXX Laboratories
Gregory L. Shipley, Ph.D., UTHSC-Houston
5:30 Networking Reception
6:30 Close of Day