|2:00-5:00 Short Course hosted by
|Getting the Most out of Your qPCR Experiments
- Review of critical assay design
- Optimization of assay
This will be an interactive workshop - attendees are encouraged to bring data files and problem examples with
Who Should Attend &
All levels of qPCR users are encouraged to participate, you will learn good laboratory practices to help you optimize your qPCR assays and effectively troubleshootyour results.
* Separate Registration Required
5:30-6:30 Early Conference Registration
Monday, October 15
8:00-9:00 am Conference Registration and Morning Coffee
9:00 Chairperson’s Opening Remarks
9:10 New Real-time PCR Technology, Mediated by Multi-Component Nucleic Acid Enzymes (MNAzymes); has a Superior Capacity for Multiplex Analysis
Elisa Mokany, Ph.D, Scientist, Diagnostics, Johnson & Johnson Research
Novel non-protein enzymes known as “MNAzymes” were exploited to develop a new real-time PCR technology with multiple advantages over other real-time chemistries. MNAzymes are composed of two short DNA oligonucleotide “partzymes” which combine to form catalytically active DNA enzymes only in the presence of target amplicons. The enzymatic MNAzyme complexes then cleave generic oligonucleotide probes between fluorophore and quencher dyes generating a fluorescent signal. Since the MNAzyme approach requires target-specific binding of two partzymes and two PCR primers, it has four levels of specificity. This is greater than that of the TaqMan and Molecular Beacon methods, which have only three levels of specificity, conferred by two primers and one target-specific probe. The use of generic probes over target-specific probes reduces the cost of assays due to scale of manufacture, ease of QA and elimination of probe waste, since unused probe can be used for new targets. Further, generic probes give reliable, consistent performance when coupled to any target or used for any PCR application. A series of five generic probes have been successfully used in a one-step RTPCR quintuplex assay that allowed simultaneous quantification of five target transcripts. The same probes were used in real-time assays for RNA or DNA quantification, analysis of methylation patterns and the detection of single nucleotide polymorphisms. The technology is highly precise as evidenced by low inter- and intra-assay variation and it has performed well on all five of the real time platforms on which it has been tested. MNAzyme PCR is a new approach to real-time PCR that allows target-specific interrogation of amplicons while using generic probes. It has the advantages of greater specificity, reduced cost, reduced time for development of assays for new targets and is much more amenable than other technologies to the development of multiplex assays including quintuplex assays.
9:40 Validation and Standardization of Quantitative Gene Expression Analysis for Microarray and Real-Time Quantitative PCR Using Universal External RNA Controls
Z. Lewis Liu, Ph.D, Research Molecular Biologist, National Center for Agricultural Utilization Research, USDA-ARS
This presentation will introduce newly developed universal external RNA controls and their applications on different platforms of microarray and real-time quantitative PCR including SYBR Green and TaqMan-probe based chemistries. Data obtained from different platforms will be presented as an example to demonstrate uniqueness of the control, and data comparability derived from microarray and qRT-PCR using the same source of RNA and the universal controls. Application of the universal controls has changed the conventional practice of quantitative PCR and made it possible as a high-throughput assay platform for gene expression analysis. The universal control is the first of its kind and filled the gap of lacking a reliable, independent, and robust standard for quantitative gene expression analysis using different platforms.
10:10 Technology Spotlight
10:25 Networking Coffee Break
10:45 TRAC in Screening of Gene Expressions in Cell Lysates
Jari Rautio, M.Sc, Biotechnology, VTT
TRAC is a novel technology enabling high-throughput gene expression analysis. Efficiency of the TRAC technology results from multiplex target detection (up to 60-plex), fast hybridisation in solution, direct use of cell lysates as sample material and automated sample processing. The proven applications of the technology range from microbial detection and bioprocess monitoring to screening of marker gene expression levels in cancer cell cultures.
11:15 Validation Steps Required for ChIP-DSL Promoter Array Development & Deciphering of Novel Biological Pathways
Jeffrey Falk, Ph.D, Director, Technology Applications, Molecular Biology, Aviva Systems Biology
A detailed account will be provided on the qPCR validation steps that are necessary for analyzing ChIP-on-ChIP data in order to decipher important biological pathways and networks. ChIP-on-ChIP is a rapidly emerging technology for mapping transcriptional regulation of important biological pathways and networks. We have developed the next generation in ChIP-Chip technology, a novel promoter array technology, ChIP-DSL (Chromatin Immunoprecipitation DNA Selection and Ligation) that results in significantly increased levels of specificity and sensitivity in identifying important Transcription Factor/promoter interactions, Epigenetic Modifications, & DNA Methylation sites. The proposed presentation will provide a step-by-step account of the detailed data analysis and validation processes required for these studies, from oligo design, to stepwise experimental quality control validation steps, to validation of the resulting array. Insight will also be provided on analyzing the resulting ChIP-DSL data, the qPCR validation steps that are necessary.
11:45 Panel Discussion with Morning Speakers
|12:15 pm Luncheon Technology Workshop
|| Sponsored by
2:00 Chairperson’s Remarks
2:05 Systematic Exploration of DNA Copy Numbers and Gene Expression using Microarrays and qPCR
Herbert Auer, Director of the Functional Genomics Core, Institute for Research in Biomedicine
Variation in gene copy numbers is a newly discovered factor contributing to a large extend to genetic variations in human populations and disease. The consequences for variation in gene expression are far from clear. We present the newly developed method of “gene resolution analysis of copy number variation” (graCNV), allowing DNA copy number measurement and gene expression using the same platform. This method generates a cohesive picture of genetic variation and phenotypic consequences where DNA and RNA measurements are easily analyzed using a single array platform; graCNV shows a high degree of validation by quantitative PCR.
2:35 Biomarker Identification Using Comparative Genomic Hybridization
Dimitri Semizarov, Ph.D., Group Leader, Tumor Genomics, Cancer Discovery, Abbott
Real-Time PCR Assessment of siRNA Based Gene Silencing
Jo Vandesompele, Ph.D., Center for Medical Genetics, Ghent
3:20 Networking Refreshment Break, Poster and Exhibit Viewing
4:00 Detection and Characterization of Copy Number Variation in the Human Genome Using Array-Based Comparative Genomic Hybridization
George H. Perry, Ph.D., Department of Pathology, Brigham & Women’s Hospital
Using multiple array-based comparative genomic hybridization (aCGH) platforms, we have recently shown that copy number variation (CNV) is surprisingly common among normal humans. This discovery has led to the consideration of CNVs in disease association studies, and sparked intense speculation that CNVs may have played important roles in human evolution, although few studies have yet tested evolutionary hypotheses for CNVs. To provide a specific example, I present the results from a study of a multi-allelic CNV, the salivary amylase gene (AMY1), which encodes for an enzyme responsible for starch hydrolysis. We found that AMY1 gene copy number correlates positively with salivary amylase protein levels, and that individuals from populations with high-starch diets have higher AMY1 copy numbers than those with traditionally low-starch diets. Comparisons with other CNV loci, using data from a whole genome tiling path aCGH platform, suggest that the observed AMY1 copy number differentiation is highly unusual; this is one of the first examples of positive selection on a human CNV.
|4:30 Real-time Quantitative PCR as an Alternative to Southern Blot or Fluorescence in Situ Hybridization for Detection of Gene Copy Number Changes
Jo Vandesompele, Ph.D., Center for Medical Genetics,
Ghent University Hospital
5:00 SNPs: Better than Gene Expression Data?
David Moskowitz, M.D., CEO, GenoMed, Inc.
Genes expressed by diseased tissue (tumors, arthritic joints, etc.) represent the last stage in a process that began decades earlier. Biological processes are famous for involving cascades with large amplification loops. A single kinase protein, for example, can activate hundreds of downstream targets. Furthermore, inhibitors rarely achieve >90% efficacy. The best hope for reducing flux through a disease pathway is to block the earliest rate-limiting steps in that pathway. Blocking late steps imperfectly will have little clinical effect. Microarrays cannot reveal the rate-limiting steps which initiate a disease pathway, which alone are the best targets for pharmacological intervention. Only genomic polymorphisms, especially single nucleotide polymorphisms (SNPs), can do that.
5:30 Panel Discussion with Afternoon Speakers
6:00 Networking Reception in Exhibit Hall
7:00 Close of Day