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Day 2
Tuesday, March 21
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8:00 Breakfast Workshop |
sponsored by |
Enhancing Sensitivity and Efficiency of Reverse Transcription
Dr. Ian Kavanagh, R&D Manager, ABgene
We have investigated the effect of primer choice, RTase enzyme and RT enhancers on sensitivity and efficiency of reverse transcription. ABgene have developed a novel RT Enhancer to remove DNA contamination, improving the sensitivity and accuracy of the subsequent
qPCR. Additionally, we have optimised a blend of random hexamers and anchored oligo dT for maximum sensitivity and versatility. |
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8:45 Chair's Remarks
8:50 Identification of Allelic Deletions with Quantitative
PCR-Based Assay
Ms. Sonia Benhamed, R&D Specialist, R&D, GeneDx Inc.
Molecular diagnostics represents an important aspect of the practice of clinical genetics. Mutation detection provides a key to the correct diagnosis and is indispensable in prenatal genetic testing. Complete deletion or whole exon deletion involving only one allele, a subset of disease-causing mutation, can only be detected by means of a quantitative assay. CopyDx is an approach to identifying allelic deletions, based on comparative quantitative real-time
PCR. Using this assay in the clinical setting, we have successfully identified whole gene deletions, single and
multi-exon deletions, and duplications underlying severe Mendelian inherited disorders in affected individuals, as well as determined carrier status in unaffected relatives at-risk of having affected offspring.
9:10 Functional Validation of "Historical" Gene Expression Data: Enabling
qPCR-Based Discovery using Binary and Multi-Component Learning Approaches
Dr. Andrew I. Brooks, Associate Professor of Environmental Medicine and Genetics; Director, Bionomics Research and Technology Center
(BRTC), Environmental and Occupational Health Science Institute, University of Medicine and Dentistry of New Jersey
Quantitative PCR has become the technology of choice in the gene expression discovery process and is touted as the gold standard for microarray validation. More recently, many basic and clinical scientists have adopted the use of qPCR for the rapid, reproducible and sensitive detection of pre-validated targets selected via a variety of higher throughput modalities. This process has shown to be successful for biological perturbations and disease states that are more "easily" classified using conventional means for target identification. To this end, very few technologies and informatics tools have been developed to address more complex, multi component disease processes preventing the use of qPCR in the diagnostic (and prognostic) arena for complex disease states. In order to enable this process both improved sample processing and data analysis tools have been developed. Coupling whole transcriptome amplification with supervised learning approaches our group has developed a technological and analytical strategy for using qPCR to validate sample fidelity, array performance, target selection and high-throughput sample screening programs for complex disease states. This approach has been applied in several biomedical areas including and not limited to: cancer biology, cardiovascular biology and neurobiology. Lastly, this approach will further enable the use of public microarray data sources by providing efficient means for target selection and validation in clinically relevant areas where limiting samples predominate.
9:40 Real-Time Reverse Transcription PCR and the Detection of Occult Disease in Colorectal Cancer
Dr. Stephen A. Bustin, Professor, Centre for Academic Surgery, Institute of Cell and Molecular Science,Queen Mary's School of Medicine and Dentistry, University of London
Molecular diagnostics offers the promise of providing an accurate match between patient and treatment, with a resultant significant effect on improved disease outcome. More specifically, the real-time reverse transcription polymerase chain reaction
(qRT-PCR), with its combination of conceptual simplicity andtechnical utility, has the potential to become a valuable analytical tool for the detection of mRNA targets from tissue biopsies and body fluids. Its potential is particularly promising in cancer patients, both as a prognostic assay and for monitoring response to therapy. Colorectal cancer provides an instructive paradigm for the potential as well as the problems associated with its use as a clinical assay. This presentation focuses on the qRT-PCR assay's potential for generating quantitative results that are not only more informative than qualitative data, but contribute to assay standardization and quality management. However, although it is evident that qRT-PCR assays have become a useful and important technology in the clinical diagnostic laboratory, it must be used appropriately and it is essential to be aware of its limitations, both technical and conceptual, if it is to fulfill its potential.
10:10 Coffee Break, Poster and Exhibit Viewing
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11:00 Molecular & Diagnostics Core Managers Roundtable Discussion
Moderator
Dr. Christian Leutenegger, Director, Lucy Whittier Molecular & Diagnostic Core Facility, UC Davis
Participants
Dr. Andrew I. Brooks, University of Medicine and Dentistry of New Jersey
Dr. Kevin Knudtson, University of Iowa
Dr. Mary Petrowski, Exelixis Pharmaceuticals
Dr. Gregory L. Shipley, UTHSC-Houston
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12:00-2:00 Luncheon Solutions Showcases
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12:00-12:15 Enabling Multiplex Real-Time PCR Technology: |
Sponsored by |
A Novel Approach To Overcome Multiplexing Limitations
Dr. Dirk Loeffert, Director of R&D, QIAGEN GmbH
Multiplex real-time qPCR adds a new level of reliability to gene
expression analysis: Co-amplification of transcripts with internal
controls minimizes variability of quantitative results, saves precious
sample material and increases throughput of real-time PCR assays. However,
multiplex real-time PCR usually is compromised by poor assay sensitivity
and linearity. The optimization of assays that is frequently required
renders this method into a tedious and costly approach. A novel technology
concept allows for the first time to employ multiplex real-time PCR in a
higher throughput mode for routine 2step- and 1step RT-PCR gene expression
analysis without the need for additional assay optimization. The
presentation will focus on the technology concept and proof data from
researchers in pharma and professional markets. |
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12:15-12:30 QZymeTM
MPX, a Multicolor SYBR Alternative
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Sponsored by |
Dr. Brad Scherer, Associate Director/Group Leader, Clontech
We have developed a novel quantitative PCR technology based on the use of a DNAzyme and a dual-labeled fluorophore universal substrate. Since all that is required are end-user supplied gene-specific primers along with the QZymeTM MPX kit, singleplex or duplex qPCR experiment can quickly and simply be done with little to no need for optimization. |
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| 12:30-12:45 Crafting Fluorescent Oligos through the Web with
RealTimeDesignTM |
Sponsored by |
Mr. Ben Sowers, Research Associate, Biosearch Technologies, Inc.
In a significant transformation of traditional PCR, real- time PCR reveals the amplicon through an accumulating fluorescent signal. Emerging from this evolution are a diverse assortment of methods to probe DNA targets, all using fluorescent-labeled
oligonucleotides. To assist with the technical task of selecting and crafting these oligo sequences, a new software program titled RealTimeDesign is presented for use over the internet. A tutorial will be provided, demonstrating the software’s rapid design of TaqManÒ
and AmplifluorÒ
assays. By submitting a panel of human genes to the software’s scrutiny, we demonstrate that RealTimeDesign proposes robust assays without additional user expertise, achieving an average amplification efficiency of 99% across seven orders of magnitude of starting copy number. By labeling the probes with spectrally distinct fluorescent reporters, we show that assay designs can be successfully multiplexed in combination with one another. These results also provide an illuminating side-by-side comparison of the TaqMan and Amplifluor probing methodologies. |
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| 12:45-1:00 Environmental Support for qPCR by Neowater™ |
Sponsored by
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Mr. Eran Gabbai, President & CTO, Do-Coop Technologies Ltd
Neowater™ is an enabling technology that is based on breakthrough water-based
nanotechnology. Unlike traditional, dry nanotechnology, which focuses on a nanoparticle end product, Neowater™ builds upon the unique properties of nanoparticles to modify the physical properties of water molecules around them and serve as better molecular environment for
PCR.
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2:00 Chair's Remarks
2:05 Global RNAi Phenotype Analysis for
Cancer Drug Target Identification and Validation by Quantitative Real-Time PCR
Dr. Sukru Tuzmen, Investigator, Head of Molecular Genetics, Cancer Drug
Development Laboratories (CDDL), Translational Genomics Research Institute (TGen)
Quantitative real-time PCR (qRT-PCR) is a widely used technique for accurate
evaluation of RNA expression levels. Due to its simplicity, wide dynamic range
of quantification, sensitivity, and precision, qRT-PCR is the method of choice
for validation of RNAi knockdowns. We describe here a global RNAi phenotype
analysis for cancer drug target identification and validation of drug targets by
qRT-PCR. To better understand the onco-selective dependencies that arise during
the neoplastic process, we have developed and applied global RNAi profiling to
discover context dependent vulnerabilities in cancer cells. Using qRT-PCR and a
functional pharmacogenomic screen for genes that affect cancer chemotherapy
response, we were able to identify potential functionally relevant genes that
could give us a better understanding of the mechanism of drug action.
Additionally, this approach can be applied to the identification of functionally
relevant candidate markers for predicting drug response, and candidate drug
targets for combination therapy to enhance the response to common cancer drugs.
2:35 The Use of Quantitative PCR in Target
Discovery, Compound Selection and Marker Development
Dr. Paul E. Kroeger, Senior Group Leader, Gene Expression Analysis, Global
Pharmaceutical Discovery, Abbott Laboratories
We use a variety of gene expression methods to facilitate the drug discovery process, and quantitative PCR is an integral tool in this effort. From the initial target discovery experiments to late stage compound optimization, gene expression signatures are used to measure and assess progress. To highlight this process a number of qPCR applications will be described including: the benefit of target distribution analysis across tissues and cells from multiple species; how differential expression of targets due to splicing is examined; how compound profiles generated with high density microarray analysis are confirmed and developed into marker panels. In summary, this presentation will describe how we concentrate on the timely and appropriate application of a collection of approaches, including
qPCR, to accelerate the drug discovery process.
3:05 Refreshment Break
(Last Chance for Poster and Exhibit Viewing)
3:30 Validation of a Target Regulating the
Expression of Key Disease Related Genes by Microarray, q-PCR and Protein-Based
Assays
Ms. Sunita Badola, Manager II, Molecular Technology, Millennium
Pharmaceuticals
There is no doubt that gene expression studies based on evaluating mRNA levels
for entire genome in diseased tissues have had a major impact on our
understanding of the molecular mechanisms underlying various diseases. Although
microarray technology has evolved over the years, the validation remains a
critical requirement. This talk will discuss some case studies where global
cellular responses in vitro has provided new insights into the gene regulation
involved in cytokine signaling.
4:00 Speaker To be Announced
4:30 Closing Panel of Experts
Dr. Bob Rutledge, Senior Scientist, CFS, Natural Resources Canada |
Sponsored by |
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5:30 Close of Conference
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