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Quantitative Real-Time PCR for Molecular Diagnostics - Day 1

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12:00 pm Conference Registration

1:30 Chairperson’s Opening Remarks

1:40 Finicky and Sloppy Molecular Beacons

Fred Russell Kramer, Ph.D., Professor, Department of Microbiology and Molecular Genetics, New Jersey Medical School

Molecular beacons are hairpin-shaped oligonucleotide probes that undergo a fluorogenic conformational change upon binding to their target. They can be labeled with differently colored fluorophores, en-abling multiplex assays to be carried out; and they can be designed to be either extraordinarily specific for the direct identification of a target sequence, or to be mismatch-tolerant for the identification of a target sequence by determination of the stability of the resulting hybrid.

2:10 Multivolume SlipChip for Quantification of Viral Load by Using Multiplex Digital RT-PCR

Feng Shen, Ph.D., Director, Research and Development, SlipChip LLC

Multiplex digital RT-PCR with large dynamic range has been demonstrated on a multivolume SlipChip for quantitative analysis of nucleic acid, specifically HIV and HCV RNA. The results of viral load test on the SlipChip were self-consistent, and the HIV viral load results of clinical patients’ samples on the SlipChip were in good agreement with results determined by Roche COBAS AmpliPrep/COBAS Tagmen HIV-1 test.

2:40 Toward HER2 PCR Testing Standardization

Kung Chiu, President, Genetics Development Corporation

Quantitative real-time polymerase chain reaction (qRT-PCR) is a relatively new technology used to quantify gene expression level in different cells or tissues. Many investigators have explored the potential of using qRT-PCR method to measure Her2 expression level in cancer tumor samples, and demonstrated that qRT-PCR is a simple, reliable and accurate method. However, a current challenge is the lack of a standard RT-PCR protocol and criteria for identification of Her2 positive cancer patients. Here, we review several published PCR HER2 testing methods and suggest a way, based on our pre-clinical trial experience, toward the standardization of qRT-PCR method in Her2 diagnostics.

3:10 Refreshment Break in the Exhibit Hall with Poster Viewing

4:00 2D-RT-qPCR and Its Application to Tissue Microarrays

Michael Armani, Ph.D., Mechanical Engineering, University of Maryland; Postdoctoral Research Assistant, Lab of Pathology
National Cancer Institute

2D-RT-qPCR is a novel technique for performing tissue lysis, RNA purification, and RT-qPCR across a tissue section.  2D-RT-qPCR was applied to frozen tissues to preserve coarse 2D architecture.  It was also applied, in conjunction with the Fluidigm system, to formalin-fixed tissue microarrays to demonstrate the possibility of a high throughput system for mRNA-based cancer molecular diagnostics. 

4:30 qPCR Quantitation of siRNA and microRNA Sequences in Animal Tissues – Challenges and Solutions

Jessica Seitzer, Ph.D., Research Biologist, RNAi Therapeutics Delivery Biology, Merck & Co., Inc.

Stem-loop PCR is a sensitive and precise methodology for measuring short nucleotide sequences such as miRNA and siRNA. Sample preparation and biological matrix have a large impact on the quantitation of miRNA and chemically-stabilized siRNA. The presentation will focus on sample preparation optimization and the significance of data interpretation on siRNA delivery.  In addition we will explore an important application of the method - using tissue-specific microRNAs as toxicity biomarkers for siRNA-based experimental therapeutics.

5:00 A Single Reference Gene is Sufficient for Normalization:  PGK1 as a Reference Gene for Quantitative Gene Expression Measurements in Human Blood RNA

Virginia Rebecca Falkenberg, Ph.D., Centers for Disease Control and Prevention (CDC)

Using the geNorm and NormFinder algorithms to select the most stable reference genes we identified Phosphoglycerate kinase 1 (PGK1) as one of the optimal normalization genes for both whole blood and PBMC RNA.  A modification of the single control normalization error (E) was used to show that a single reference gene is sufficient for normalization when the most stable candidates are used.  These findings should facilitate large-scale molecular epidemiologic studies using blood RNA as the target of gene expression measurements. 

5:30 Close of Day One

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