Monday, March 20

7:30 Registration and Morning Coffee

8:00 Chair's Opening Remarks

8:15 Kick-Off Keynote Quantitative PCR: A Convergence of Procedural, Instrumentation and Biological Issues

Dr. John J. Sninsky, Vice President of Discovery Research, Celera Diagnostics The inherent quantitative capability of PCR was appreciated early on in this powerful and protean procedure’s conceptual and implementation phases. The exponential nature of the procedure in which small differences in amplification efficiency per cycle translate into large changes after many cycles is at the heart of the concern of reproducibility and accuracy. Multiple procedural advancements, primary among them being real time PCR and the quality of thermal cycling instrumentation, have facilitated and fostered widespread use. A brief summary of critical developments will be briefly reviewed. Quantitative PCR has permitted numerous important questions to be answered and indeed permitted the posing of questions not previously thought feasible. The exquisite sensitivity has raised fundamental questions about the biological meaning of detection. Indeed questions beyond absolute levels of a targeted template at a single time point have evolved to query whether and to what extent levels of the template nucleic acid were changing over time and whether the changes had biologically or clinical implications. Further, the appreciation of the redundancy in the human and other organism genomes has raised the question of what exactly is being amplified. Primer and probe design is critical to interpretation of the resulting data. The performance of quantitative PCR has involved with convergence of procedural, instrumentation and biological issues. Quantitative PCR has transformed how we think of biological tenets and promises additional advances to further enlighten our fundamental understanding whether it be from samples in the distant past or now.

9:00 Quantitative Real-Time PCR for Detecting Viral Pathogens at a Clinical Reference Lab
Dr. Jeffery B. Stevenson, Senior Scientist, ARUP Institute for Clinical & Experimental Pathology 
Real time PCR is now commonly used for detecting pathogens in clinical samples. Many assays are currently being developed for clinical use that take advantage of the intrinsic quantitative nature of real-time PCR. We have developed and implemented real-time assays for the absolute quantitation of Human Herpesvirus-6, Epstein-Barr virus and BK virus, using hybridization probe technology. Validation data for these assays will be presented. In addition, important issues to be considered when developing an accurate and reproducible real time quantitative assay will be discussed.

9:30 A Method to Monitor Gene Expression via Direct qRT-PCR of Cytoplasmic Lysate *Patent Pending 
Dr. Carolyn Bialozynski, Senior Research Associate, Research & Development, Pierce Milwaukee, LLC
Current methods to isolate and purify RNA exhibit two inherent shortcomings. First, these methods commonly use reagents that lyse the entire cell, thereby causing the desired spliced and fully-functional cytoplasmic RNA to be in a matrix with nuclear debris that can interfere with downstream applications such as real-time quantitative RT-PCR (qRT-PCR). Second, these methods do not efficiently retain small RNA species. To address these issues, we have developed a method for effective compartmentalization of all cytoplasmic RNA species away from nuclear components. These cytoplasmic extracts can then be used directly in qRT-PCR applications with no further DNase treatment or purification. This method of compartmentalizing cytoplasmic RNA allows for direct and efficient monitoring of gene expression and short interfering RNA (siRNA)-mediated mRNA silencing via qRT-PCR, as well as quantitation of fully-functional mRNA (providing more meaningful correlation between gene and protein expression). Experiments to demonstrate effective compartmentalization and direct use of cytoplasmic lysates in real-time qRT-PCR applications will be presented.

10:00 Coffee Break

10:15 Use of Direct mRNA Quantification and Spike-In Targets to Perform Optimization of Microarray Processes
Dr. Paul Wolber, Integrating Manager, Microarray QC Development, Agilent Technologies
Microarrays have become important tools for performing parallel measurements of the relative expression levels of large numbers of genes amongst different samples. However, microarrays are known to suffer from systematic errors that diminish or "flatten" measured expression ratios below their true values. We have used the combination of external spike-in mRNA standards and a direct mRNA assay ("QuantiGene" assay) to objectively optimize both microarray production methods and sample processing methods. The optimization projects have resulted in substantial improvements in the accuracy of expression ratio measurements, and demonstrate the power of orthogonal analytic methods to guide further advances in microarray technology.

2:00 Chair's Remarks

2:05 PCR-Based Method for Quantitation of Attomolar Levels of microRNA and Other Short Nucleic Acid Sequences
Dr. Bridget A. Lollo, Associate Director, Microrna Drug Discovery, Isis Pharmaceuticals and Consultant, Geneca Technologies 
MicroRNAs are short ~22mer RNA molecules that are endogenously encoded within the genome from plants to mammals. These non-coding RNA molecules are found to have important regulatory functions in cell growth, differentiation, apoptosis and exocytosis. MicroRNAs present a unique challenge for quantitation because of their small size, the sequence similarity among family members and their occurrence in several maturational forms. The two primary approaches to quantitation have been northern analysis and various high density array-based detection systems. What is lacking in these methods is the ability to accurately quantify microRNA in a high-throughput fashion in small amounts of RNA. Here we describe a sensitive technique to quantify microRNA in total RNA samples that can distinguish among family members and immature forms and correlates very well with northern quantification. Biological applications using this technique, which we call quantitative PCR for short RNA, or SQ-PCR, will be shown.

2:35 A New Method for Amplification of RNA from Clinical Samples for Gene Expression Analysis
Dr. Paul D. Siebert, Director of Research, System Biosciences (SBI) 
Due to the poor quality of the resulting RNA, gene expression of RNA obtained from clinical samples has been problematic. RNA obtained from formalin-fixed paraffin-embedded tissue (FFPE), for example, is chemically modified and can be highly degraded. Here we present a new method for amplification of RNA from FFPE tissues that can be completed in less than 3 hours, is not biased toward the 3'-end of the mRNA, and, as opposed to previous methods, can successfully amplify degraded RNA. Through RT-PCR the method is applied to gene expression analysis. In addition, the method is ideally suited for analysis of alternatively spliced gene transcripts since nucleotide sequences all along the length of the RNA are maintained.

3:05 GeXP: A Novel, Highly Multiplexed qPCR Method
Dr. Joseph Monforte, Chief Scientific Officer, Testing & Discovery, Althea Technologies, Inc. 
The current state of the art in quantitative PCR using real-time methods has severe limitations in terms of multiplexing capability. While it is conceivable to develop 5-plexes, for gene expression analysis where quantitation needs to be highly accurate, the practical limitation is a 2-plex: one gene plus one control. So effectively real-time methods are one-gene-at-a-time methods. In partnership with Beckman Coulter, we have developed an alternative strategy that enables very high levels of quantitative, multiplexed PCR for gene expression analysis. The method called GeXP utilizes a novel, universal priming strategy and end-point PCR to multiplex 20-35 genes in a single PCR reaction and yields highly quantitative readout using fluorescence-based capillary electrophoresis systems. We will present specific data on the use of several different multiplexes as applied to molecular research and diagnostics, including immunology with human blood analysis and the diagnosis of multiple types of childhood cancer.

Technology Spotlights

3:35 Nucleic Acid Stabilization in Cultured Cell and Tissue Lysates for qPCR Gene Expression Analyses
Dr. Lee Scott Basehore, Senior Research Associate, Nucleic Acids Analysis R&D, Stratagene
A novel buffer is described for lysis of cells and stabilization of released nucleic acids. Lysed cells can be stored for 6 months before qPCR analysis is performed. RNA in stored cell lysates shows little or no degradation by Agilent Bioanalyzer analysis. RNA and DNA can be amplified from cultured cell and tissue lysates without the need for purification. The lysate contains the entire nucleic acid complement, allowing the use of single copy DNA markers to normalize input cell mass and accurately measure gene expression between samples by qPCR. 

3:50 Accurate Quantification of microRNA Expression using Real Time PCR
Dr. Michael J. Antinore, Product Manager, Nanogen

4:05 Refreshment Break, Poster and Exhibit Viewing

4:45 Venting and Validation: Focused Group Discussions
Does technology support specific applications or do applications support the development of a specific technology? Time has been designated for focused group discussions centered around a specific topic. This is a moderated discussion with brainstorming and interactive problem solving between scientists who share a common interest on the discussion topic. The interaction between those developing the technology and those utilizing the applications will produce results and develop an expanded network of fellow researchers working in your area of research interest. 

5:45-7:00 Networking Reception in Exhibit Area