MONDAY, MARCH 16
7:30 am Registration and Morning Coffee
PRE-ANALYTICAL SAMPLE PREPARATION:
EXTRACTION AND PURIFICATION
8:30 Chairperson’s Remarks
Jo Vandesompele, Ph.D., Professor, Center for Medical Genetics, Ghent University Hospital
8:40 KEYNOTE PRESENTATION
qPCR: Historical Perspectives and New Publication Guidelines
Carl Wittwer, Ph.D., Professor, Pathology, University of Utah
Quantitative polymerase chain reaction (qPCR) has become a central enabling technology in life science research and diagnostics. Originating in the 1990s, real time analysis provides a simple, homogeneous method for quantification that is rapid, sensitive and specific. Its popularity and power have led to many different instruments, reagents and protocols. MIQE is a collection of guidelines that describe the Minimum Information necessary for evaluation of Quantitative real-time polymerase chain reaction Experiments.
9:30 Reliable Measurement of Gene Expression in Formalin-Fixed Tissues Using the Branched-Chain DNA Assay
Beatrice Knudsen, M.D., Ph.D., Program in Molecular Diagnostics, Division of Public Health Sciences, Fred Hutchinson Cancer Research Center
Formalin fixation and tissue processing damage, chemically modify and crosslink RNA, significantly hindering accurate quantification. We tested the QuantiGene b-DNA assay on a panel of genes and noted remarkable accuracy, reproducibility and sensitivity compared to standard qPCR. The QuantiGene accurately reported expression differences of known cancer genes between normal and cancerous prostate tissues obtained from 8–13 years old archival tissue blocks.
10:00 Novel Solution to Old Problems: Solid Phase Gene Extraction
Karl H. Hasenstein, Ph.D., Chance Professor of Biology, Biology Department, University of Louisiana, Lafayette
Most small-scale mRNA extractions depend on the removal of cells or tissue from an organism or preserved specimens. These methods are destructive and do not distinguish between genomic DNA and RNA such that extracted (m)RNA is typically contaminated and the required purification leads to loss of especially low-abundant mRNA. The ideal removal of mRNA minimizes RNA loss, eliminates the need for reference genes, and allows repeated sampling from living material. These goals resulted in the development of Solid Phase Gene Extraction (SPGE). The hybridization of gene-specific or generic sequences to a solid surface and subsequent q-PCR allows rapid and quantitative analyses of gene expression with minimal damage to the sampled system.
10:30 Coffee Break
11:00 RNA Integrity for Bacterial RNA Quantitation
Courtney Jahn, Ph.D., Post-Doctoral Fellow, Bioagricultural Sciences and Pest Management, Colorado State University
RNA integrity is critical for successful RNA quantitation for mammalian tissues, but the level of integrity required for quantitation had not been determined for bacterial RNA. We found that RNA preparations of different quality yielded drastic differences in relative gene expression ratios and led to major errors in the quantification of transcript levels. The RNA integrity number (RIN) values below 7.0 resulted in excessive variation and loss of statistical significance when gene expression was analyzed by real-time qRT-PCR. This work provides guidelines for RNA isolation and quality assessment that will be valuable for gene expression studies in a wide range of bacteria.
11:30 Panel Discussion with Morning Speakers
12:00 Close of Morning Session
Sponsored by
12:15 Luncheon Presentation
Considerations for Speeding up qPCR Research
Ian Kavanagh, Ph.D., Research and Development Manager Genomics, Thermo Fisher Scientific
With the ever-growing popularity in the use of qPCR as a sensitive technique for quantifying DNA and RNA, there is increasing demand for higher throughput of reactions and faster protocol times. There are many steps within the workflow of a reaction that can be optimised to reduce the overall time they take. However, developing a fast QPCR assay has many challenges. In particular, the improvement in speed should not come at a loss of assay performance. Discussion points include: the common steps taken to complete a QPCR assay; steps that can be taken in order to minimize the variation throughout the protocol; and ways in which these steps can be optimised to reduce the overall time of a QPCR protocol.
The HANDYLAB Jaguar System: An Integrated Platform for Molecular Diagnostics
Sundaresh Brahmasandra, Ph.D., VP Product Development, HandyLab, Inc.
HandyLab has developed the Jaguar System comprising a proprietary sample extraction platform effortlessly integrating with a microfluidic real-time amplification module resulting in a truly self-contained, integrated and walk away solution to rapid molecular diagnostics. The Jaguar system incorporates a novel sample preparation technology in room temperature stable, pre-loaded, disposable strips to process individual samples from a wide variety of clinical specimens. Upon extraction & clean-up, the eluted nucleic acid is mixed with pre-loaded amplification reagents, loaded into a microfluidic cartridge and real-time PCR is performed. The system allows for the simultaneous operation of tests in 'closed' format using FDA-cleared assays as well as in 'open' format using Lab Developed Tests or assays using analyte-specific reagents (ASR), thereby enabling true random access operation and significantly enhancing the utility of such systems. Data demonstrating the performance, flexibility and reliability of the system will be presented in the talk.
NOVEL TECHNICAL APPROACHES
2:00 Chairperson’s Remarks
Daniel McClernon, Ph.D., Consultant, McClernon LLC
2:05 miR-Q: qRT-PCR for Expression Profiling of Small RNAs Such as MicroRNAs
Soroush Sharbati, Ph.D., Institute of Veterinary-Biochemistry, University of Berlin
The miR-Q approach is a highly sensitive quantitative reverse transcription PCR (qRT-PCR) for expression analysis of small RNAs such as microRNA molecules. It is based on SYBR Green detection without requiring fluorochromic probes or Locked Nucleic Acid (LNA)-modified oligonucleotides. It shows a high dynamic range of six to eight orders of magnitude comprising a sensitivity of up to 0.2 fM microRNA, which corresponds to single copies per cell. There is nearly no cross reaction among closely-related microRNA family members, which points to the high specificity of the assays.
2:35 Monodisperse Picoliter PCR: Adding Another Dimension to qPCR
Reginald Beer, Ph.D., Research Scientist and Principal Investigator, Lawrence Livermore National Laboratory
Quantitative PCR is a mainstay of the life sciences, impacting genomics and diagnostics to name a few. However, the method to date has not benefited from the ability of digital microfluidics to partition the PCR sample and obtain extremely accurate target concentrations compared with current methods. Research in monodisperse picoliter PCR, where the droplets function as individual isolated reactors with identical reaction rates, has shown the promise of this exciting new approach. This talk will detail multiple droplet-based architectures and their performance in detecting single-copy RNA, DNA, and virions.
3:05 A Novel Method for Successful Amplification of Extremely GC-rich Promoter Regions Using the ‘Slowdown PCR’ Technique
Ulrich Frey, M.D., Institute for Pharmacogenetics, University of Essen Medical School
PCR-amplification of GC-rich templates is often hampered by the formation of secondary structures like hairpins and higher melting
temperatures. The novel method termed ‘Slowdown PCR´ allows the successful amplification of extremely GC-rich (> 83%) targets. The protocol relies on combination of a novel standardized cycling protocol with varying temperature ramping rates plus the addition of 7-deaza-2’-deoxyguanosine, a dGTP analogue and is versatile not only for amplification of extremely GC-rich regions, but also for routine DNA diagnostics and pharmacogenetics for templates with different annealing temperatures.
3:35 The Next Generation in Hot Start PCR - CleanAmp™ dNTPs Sponsored by 
Natasha Paul, Ph.D., Senior Staff Scientist, TriLink BioTechnologies, Inc.
This presentation describes an approach in which the dNTPs, an essential PCR component, are modified with thermolabile dNTP protecting groups for application to Hot Start activation schemes in PCR. Studies describe the development of this technology, including the proof of principle experiments that identified the optimal temperature-sensitive 3’-nucleoside protecting group.
3:50 Refreshment Break
DATA MODELS FOR OPTIMIZATION
4:15 Computational Optimization of PCR Assays for DNA-Based Cancer Diagnostics
Ali Bashir, Ph.D. Student, Bioinformatics Program, University of California, San Diego
The use of DNA lesions as diagnostic markers in cancer is challenging, because tumor cells are frequently admixed with normal cells, particularly in early stage tumor samples. Detection is further confounded by the fact that the rearrangement boundaries are not conserved across individuals, and might vary over hundreds of kilobases. Here, we present an algorithm for designing PCR primers and oligonucleotide probes to assay for these variant rearrangements, which yields near optimal detection in most regions. We applied our algorithm to the highly variable CDKN2A region and succeed in experimentally detecting breakpoints across multiple cell-lines, even when the region has undergone multiple rearrangements.
4:45 Primer Approximation Multiplex PCR (PAMP): A Novel Approach to Selectively Amplify Cancer Genomic Breakpoint Sequences in The Presence of Normal Cells
Yu-Tsueng Liu, M.D., Ph.D., Assistant Professor of Medicine, Division of Infectious Diseases, Director, Biomarker Laboratory, Moores Cancer Center, University of California, San Diego
It is relatively easy to use PCR to detect a foreign virus in comparison to a cancer cell because the genome sequence of a virus is unique in a human body. Nevertheless, cancer cells acquire distinctive mutations which are not present in the normal cells during the process of clonal selection. Our novel approach to identify such unique sequences as a binary (cancer vs. normal) biomarker will be presented.
5:15 Automated, Optimized Primer Design for qRT-PCR Applications
Alison Ziesel, BS.C., Department of Ophthalmology, Emory University
Quantitative real time PCR (qRT-PCR) is a commonly used method for assaying levels of specific mRNA, and is often employed as a confirmatory experiment for other expression assays, such as microarray-based experiments. However, qRT-PCR necessitates the selection of very specific target sites within mRNA species of interest. Identification and design for these sites adds a level of complexity to primer design and can unnecessarily complicate the process, possibly even compromising the quality of the experiment. To ameliorate these issues, we have developed an open source utility that combines the power of the popular Primer3 package with an automated target site identification, chemistry conditions specification, primer design, and resultant design data organization process, called MultiPriDe (Multiple Primer Design Package).
5:45 Grand Opening of the Exhibit Hall
7:00 Close of Day