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Quantitative PCR - Day 2

 

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THURSDAY, MARCH 18, 2010

7:00 am Registration Open

7:30 Breakfast Presentation (Sponsorship Opportunity)

 

TIPS, TRICKS, AND TECHNIQUES

8:15 Chairperson’s Remarks
Linda Starr-Spires, Ph.D., Director, Global Clinical Immunology, Sanofi Pasteur

8:20 MNAzyme qPCR Technology has Superior Multiplexing Capabilities and Provides a Novel Detection Strategy for SNPs

Alison Todd, Ph.D., CSO & General Manager, SpeeDx Pty Ltd.

We have developed a new qPCR technology that utilizes novel non-protein enzymes known as “MNAzymes”. The catalytically active MNAzymes cleave generic reporter probes and generate fluorescence. The use of generic probes, as opposed to target specific probes, provides multiple advantages over other qPCR methods. A novel method for single base discrimination has been further developed and utilized for SNPs. For all applications, MNAzymes have a superior capacity for multiplex analysis, due to their robust nature and cost effectiveness.

8:50 Utilizing Cell Lysates in Real-Time qPCR

Gregory L. Shipley, Ph.D., Assistant Professor, Director, Quantitative Genomics Core Laboratory, University of Texas Health Science Center

The isolation of RNA for various real-time qPCR applications is expensive, takes time and is impractical for small cellular populations on 96- or 384-well plates. Utilizing cell lysates directly for cDNA synthesis eliminates these issues as well as any chance of skewing the RNA population. An advantage of cell lysates is that DNA can be used for data normalization. Data will be presented on using cell lysates for real-time qPCR analysis.

9:20 HRM and Sequencing a Platform for In-Depth Single Locus Methylation Studies

Tomasz K. Wojdacz, M.S., Ph.D. Candidate, Human Genetics Institute, University of Aarhus

Methylation Sensitive High Resolution Melting is a cost and labor efficient method for single locus methylation screening. Bisulfite-sequencing is a gold standard method for single locus methylation studies but is expensive and laborious. We have combined MS-HRM technology with didoxynuclotide and pyrosequencing platforms. The combination of these two platforms allows us to obtain quantitative information on both the overall methylation status of the locus and single CpG sites within the locus of interest, in a cost and labor efficient fusion.

9:50 Quantification of DNA libraries for Next Generation Sequencing  Agilent Technologies
Bernd Buehler, Ph.D., Senior Scientist, Stratagene Products Division, Agilent Technologies

10:05 Networking Coffee Break, Exhibit & Poster Viewing

10:45 Real-Time PCR for Simultaneous Multiplexed Gene Detection in Multiple Single-Cell Samples

Adrian Ozinsky, Ph.D., Scientist, Microfluidics, Institute for Systems Biology

Decoding the complexity of multicellular organisms requires analytical procedures to overcome the limitations of averaged measurements of cell populations, which restrict the ability to distinguish between the responses of individual cells within a sample. We describe a straightforward method for simultaneously measuring the expression of multiple genes in a multitude of single-cell samples, and demonstrate a detection sensitivity of 30 mRNA molecules per cell, with a fractional error of 15%. We use the method to expose unexpected heterogeneity in the expression of immune-related genes in macrophages, as well as to assess co-ordinate gene expression in cancer stem cells.

11:15 Autocatalytic Aptazymes Enable Ligand-Dependent Exponential Amplification of RNA

Bianca J. Lam, Ph.D., Departments of Chemistry & Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute

Ribozymes have been developed to undergo exponential amplification by a cross-replication cycle. By combining the catalytic domain of these ribozymes with aptamers (ligand-binding domain), the exponential growth rate of these cross-replicating aptazymes reflects the concentration of ligand present. This assay may be extended to detect a variety of small molecules and proteins.

11:45 Standardization of RT-qPCR Data from Independent Biological Replicates

Erik Willems, Ph.D., Neuroscience, Aging and Stem Cell Research Center, Burnham Institute for Medical Research

Gene expression analysis by quantitative reverse transcription PCR (qRT-PCR) allows accurate quantifications of messenger RNA (mRNA) levels over different samples. Corrective methods for different steps in the qRT-PCR reaction have been reported; however, statistical analysis and presentation of substantially variable biological repeats present problems and are often not meaningful, for example, in a biological system such as mouse embryonic stem cell differentiation. Based on a series of sequential corrections, including log transformation, mean centering, and autoscaling, we describe a robust and powerful standardization method that can be used on highly variable data sets to draw statistically reliable conclusions.

12:15 pm Close of Morning Session

12:30 Luncheon Presentation Sponsored by ThermoScientific
Choosing the Correct qPCR Assay:  Performance and Research Applications
Melissa Kelley, Research Scientist, Thermo Fisher Scientific
qPCR is performed in every segment of life science research; utilizing high performance assays is essential for generating high confidence data.  Solaris™ qPCR Gene Expression Assays are ideal for routine molecular applications because they combine minor groove binder (MGB) and Superbase technologies with a rigorous design algorithm for detection of all known splice variants of a gene target, while distinguishing among closely related family members. Here we describe an application of this new probe-based detection technology in a study of the highly related AKT gene family members in regulating the activation of FOXO1 transcription factor.
 

 

 

SAMPLE PREPARATION:
EXTRACTION & PURIFICATION

2:00 Chairperson’s Remarks
Gregory L. Shipley, Ph.D., Assistant Professor, Director, Quantitative Genomics Core Laboratory, University of Texas Health Science Center

2:05 FEATURED SPEAKER
DNA Extraction from Human Stool, Primer Design, Assay Optimization

Stephen Bustin, Ph.D., Professor, Molecular Science, Barts and the London School of Medicine and Dentistry

2:35 A Tumor Sorting Protocol that Enables Enrichment of Pancreatic Adenocarcinoma Cells and Facilitation of Genetic Analyses

Zachary Boyd, M.S., Senior Research Associate, Development Oncology Diagnostics, Genentech, Inc.


Sponsored by

Integromics

 

3:05 RT-PCR Data Analysis: Avoiding Pitfalls
Alexandra Vamvakidou-Thomas, Product Manager, Integromics
With the advances in RT-qPCR technology, challenges to obtain reliable results have become more apparent due to pitfalls related to data analysis.  An experimental design is essential to the success of a study and must be done prior to sample preparation. Yet the most common pitfall is an inadequate experimental design that lacks a proper number of replicates, especially in a large project. Statistical inferences cannot be made with a limited sample size.In most experimental projects, quality control is thought of as an optional step and is often skipped. That leads to the second pitfall: how confident are we in the results, even when we are convinced that we have followed protocol? In reality, quality control should always be considered mandatory in order to ensure reliable results.  Regular check points should be set during sample preparation, processing and data analysis to reduce variance and ensure each time- reproducible results. It is critical to ensure the credibility of our conclusions by carefully tracking the analysis steps. Frequently, it becomes impossible for an independent group to reproduce the same results using the same data - reducing the consistency and impact of the results. During the analysis, a strict workflow should be followed to minimize the discretional criteria and input of humans in the process.  In conclusion, scientific data analysis workflows are important to form a structured process that produces reliable and reproducible results. The presentation will review the entire workflow process of PCR data analysis and how this is achieved using RealTime StatMiner®.

 

3:30 Networking Refreshment Break, Poster Viewing & Final Exhibit Viewing
 
4:00 Methylation of Human Papillomavirus DNA as Marker of the Progression of Cervical Cancer

Hans-Ulrich Bernard, Ph.D., Professor, Molecular Biology & Biochemistry, University of California, Irvine

The diagnosis of cervical cancer by the Pap test is rapidly becoming amended or even replaced by HPV DNA testing. HPV DNA testing, commercially available (e.g. by the Hybrid Capture II test), is sensitive but does not distinguish between asymptomatic and neoplastically progressing infections. Cancer progression goes along with recombination of the HPV DNA with the cellular DNA, and the consequence of this recombination is an extensive methylation of the HPV DNA. We are characterizing the emerging methylation patterns and their linkage to pathology by a variety of PCR and bisulfite modification based techniques, including efforts of quantifying mixtures of methylated and unmethylated HPV DNA.

4:30 Quantitative Analysis of Multiple Gene Expressions in a Single Cell by q-PCR without Pre-amplification

Hideki Kambara, Ph.D., Hitachi Central Research Laboratory

A cDNA library produced from a single-cell is directly and repeatedly used for analyzing multiple gene expressions quantitatively by q-PCR. cDNA molecules as small as several copies can be successfully analyzed with the method.

5:00 Application of Digital Microfluidics to Point-of-Care Diagnostics

Michael Pollack, Ph.D., Co-Founder, Advanced Liquid Logic, Inc.

Digital microfluidics, characterized by precise, programmable manipulation of liquid droplets using electrowetting, enables complex analytical protocols to be implemented in a low-cost, rapid and portable format. When combined with integrated sample preparation capability, digital microfluidics can serve as a broadly useful platform for point-of-care applications.

5:30 pm Close of Day