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Wednesday, April 26
7:30-8:30am Registration and Morning Coffee
Opening Plenary Session
| Keynote Presentations
8:30 Chairperson's Opening Remarks
8:40 Probing Genomes, Pathways and Disease using Microarrays
Dr. Michael P. Snyder, Lewis B. Cullman Professor and Chair, Molecular and Cellular Developmental Biology Department, Yale University
We have been using DNA titling arrays to identify transcribed regions and regulatory elements throughout the human genome. Protein microarrays are used to probe biochemical activities and posttranslational modification networks and human disease.
9:20 'The 39 Steps' in Gene Expression Profiling: Deciphering Cellular States of Innate Tumor Drug Responses
Dr. Charles Auffray, Research Director, CNRS; Genexpress, Functional Genomics and Systems Biology for Health, Pierre et Marie Curie University
Microarray technology is in a relatively immature stage compared to sequencing, with important limitations in the production of the accurate and extensive data required for integrative systems biology approaches to the complexity of living systems. To realize the full potential of microarray technology, and provide meaningful insights into the behavior of the biological systems investigated, it is essential to develop and implement standards at each and all steps of the process. These include careful study design, controlled annotation of resources and extensive quality control of experiments with associated quality metrics, the use of robust statistics including power analysis for hypothesis testing, and data description, registration and storage in public repositories using ontologies and controlled vocabularies. I will review the 39 key steps we found essential to establishing a generic quality assurance pipeline for gene expression profiling, using transcriptome analysis to decipher cellular states of innate tumor drug responses as a case study. I will highlight the potential of microarrays to uncover molecular signatures of diagnostic value, to provide a rich source of working hypotheses on underlying functional and regulatory networks, and to help identify targets for therapeutic intervention. |
10:00 Coffee Break
RNA Stability
10:30 Chairperson's Remarks
10:35 Biobanking of Fresh Frozen Tissue: RNA is Stable in Non-Fixed Surgical Specimens
Dr. Johan Botling, Genetics & Pathology, Uppsala University
The quality of RNA is of central importance for gene expression analysis of laser microdissected tissue specimens. In order to investigate the influence of tissue procurement procedures different storage times and transport media were tested systematically. Immediately after surgery, tonsil and colon tissue was cut into pieces and either snap frozen (time point 0) or (i) kept at room temperature, (ii) on ice, (iii) in normal saline or (iv) in a commercial RNA-stabilizing buffer (RNAlater) for 0.5 h, 3 h, 6 h and 16 h. RNA integrity was analyzed by microchip electrophoresis. Additionally gene expression HIF1a, PCNA, Bcl2, c-fos, TGFβ1 and SMAD7 was analyzed under these conditions using real-time PCR. Our results indicate that routine storage on ice until final freezing well preserves the RNA integrity and expression levels of representative genes in tissue specimens collected in clinical routine.
11:05 Assessment of RNA Quality for Gene Expression Analysis
Dr. Andreas Missel, Associate Director, R&D, QIAGEN GmbH
Pre-analytical steps contribute significantly to the outcome of gene expression analyses. Specific and nonspecific degradation of cellular RNA introduce changes to the transcriptome prior to analysis. Thus, assessment of RNA quality and integrity is an indispensable prerequisite for all downstream applications in transcriptome analysis. We will discuss various approaches to determine the suitability of RNA samples for applications such as microarray analysis and real-time
PCR. |
Sponsored by
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11:35 Assessment of Fixatives, Fixation and Tissue Processing on Morphology and RNA Integrity
Dr. Melissa L. Cox, Postdoctoral Fellow, Pfizer Global Research and Development
12:05 Lunch on Your Own
1:15 Technology Workshop
(Sponsorship Available)
Build your own sundae and learn what new technologies have to offer.
2:00 Chairperson's Remarks
RNA Quality
2:05 RNA Extraction from Difficult Samples
Dr. Marianna M. Goldrick, Senior Scientist, R&D, Ambion, Inc
As the only readily accessible and expendable normal tissue, blood is highly desirable for use in biomarker discovery. However the high RNase content and low concentration of nucleated cells make blood a challenging type of sample for RNA extraction. Several novel approaches have been developed recently to facilitate gene expression studies from human and mouse blood. Methods have also been developed for extraction of high yields of high-quality total RNA, including microRNA, from whole blood and fractionated WBCs. Results of initial studies will be presented, comparing blood microRNA profiles between healthy humans and between several inbred strains of mice. |
Sponsored by
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2:35 The qTIAM Method is an Accurate Predictor of mRNA Integrity
Dr. Eric Fedyk, Senior Scientist II, Discovery Technologies, Millennium Pharmaceuticals, Inc.
The integrity of mRNA transcripts is a principle component of the quality of transcriptional profiling data. Quantitative PCR, transcript integrity assays of mRNAs (qTIAMs), were designed to measure the quality of RNA samples, in efforts to improve sample selection (QC assay) prior to downstream assays and to assist analysis of expression data. Each of four microarray-based metrics of transcript integrity correlated more highly with qTIAM data than with the 28S / 18S ratio or the RNA Integrity Number(RIN). qTIAM data were also more accurate than RIN data, at predicting the technical quality of hybridization data; qTIAM models consistently predicted 2- to 3-fold fewer false negatives (high quality samples incorrectly classified as low quality) than RIN-based, predictive models. Hence, qTIAMs are extremely valuable QC assays for guiding selection of samples and assisting analyses of TxP datasets.
3:05 Panel Discussion: The Chicken or the Egg: Standardization vs. Quality
3:55 Refreshment Break, Poster and Exhibit Viewing
Closing Plenary Session
| Featured Presentations
4:15 Chairperson's Remarks
4:20 Functional Genomics and Proteomics Driving Individualized Medicine
Dr. Towia A. Libermann, Associate Professor of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School; Director, BIDMC
Genomics Center, Dana Farber/Harvard Cancer Center Cancer Proteomics Core
Subtle genetic differences between individuals combined with environmental effects result in distinct
predisposition to and development of diseases as well as divergent responses to therapies. Patients with the same disease respond differently to drugs due to individual differences in the particular disease causing mechanisms. Similarly, human beings respond differently to drugs due to individual variations in drug metabolism. Functional genomics promises the design of therapies optimally tailored to each patient based on individualized molecular characterization of the patient's disease process and drug response, leading to more effective treatments and less adverse reactions. Examples of genomics and proteomics approaches to stratify patients based on their individual disease mechanism and to apply individualized, more targeted and more specific therapies will be illustrated.
4:55 Molecular Network Analysis using Reverse Phase Protein Microarrays: Towards Routine Clinical Use for Patient Tailored Medicine
Dr. Lance A. Liotta, Professor and Co-Director, Center for Applied Proteomics and Molecular Medicine, George Mason University |
5:30 Networking Reception
6:30 End of Day One
Thursday, April 27
7:30 Morning Coffee (Breakfast Technology Workshop Sponsorship Available)
Opening Plenary Session
| KEYNOTE PRESENTATIONS
8:30 Chairperson's Remarks
8:40 Improving the Efficiency and Reliability of Microarray Research to Study Prognosis and Response to Therapy
Dr. David F. Ransohoff, Professor of Medicine & Director, Clinical Research Curriculum, University of North Carolina-Chapel Hill
Hundreds of papers have been published reporting that transcriptional profiling, using microarrays, may be used to predict cancer prognosis and response to therapy. Yet recent critiques suggest that many results may not be reproducible. What may be wrong with the process of evaluating molecular markers for cancer, and how can the efficiency and reliability of the process be improved?
9:20 Array CGH in Clinical Pathology: Progress and Challenges
Dr. Shelly R. Gunn, Instructor, Cellular & Structural Biology, University of Texas Health Science Center at San Antonio and
Dr. Mansoor S. Mohammed, Director, Advanced Technologies, Quest Diagnostics and Nichols Institute
Array CGH began as an effective laboratory tool for genome scanning. It is now poised to become one of the most powerful diagnostic and prognostic tests in clinical pathology. Great progress has been made in the development of clinical diagnostic arrays for congenital abnormalities, and CGH arrays are also being developed to provide detailed outlines of hematopoietic and solid tumors. However, despite this progress there are still many challenges to be met before array CGH becomes a routinely ordered clinical test. |
10:00 Morning Coffee, Poster and Exhibit Viewing
Quality RNA from Tissues
10:45 Chairperson's Remarks
10:50 Expression Microarrays from Formalin Fixed Paraffin Embedded Tissue, Considerations in the Reduction to Practice
Dr. Stephen M. Hewitt, Chief, TARP Lab, NCI/NIH
There is great interest in the introduction of expression microarrays to routine clinical practice. The most common material available for extraction of RNA is formalin fixed paraffin embedded tissue. Obtaining RNA from archival blocks is possible; however the approach requires optimization at multiple steps in the process. There are issues of specimen handling from the patient till RNA extraction that impact RNA quality. Defining the quality of RNA obtained from archival tissue is challenging. Array design can be anticipated to differ from current designs. Finally, interpretation must be considered. This talk will discuss the issues that have been identified and attempt to define what is known and what remains to be discovered as expression microarrays from formalin fixed paraffin embedded tissue become a tool for clinical medicine.
11:20 Molecular Profiling of Clinical Specimens with Degraded RNA/DNA
Dr. Richard Everson, Professor, Department, Karmanos Cancer Institute, Wayne State University
High-Throughput, highly-multiplexed assays for molecular profiling of clinical specimens with degraded RNA or DNA will be discussed. Major focus will be on assays for gene expression using formalin-fixed, paraffin-embedded clinical samples, and their use as diagnostic and prognostic factors in cancer research.
11:50 High-Quality Microarrays with RNA from FFPE Samples
Dr. Guido Krupp, CEO & President, AmpTec GmbH
Apart from RNA quantity, RNA from FFPE samples suffers from two major quality problems: cross-linking and degradation. Both issues result in very limited mRNA sequence availability for mRNA amplification and hence microarray hybridization. A new approach is presented, that permits specific mRNA amplification, even from mRNA fragments without any poly(A) tail. Data will be presented, comparing microarray data (using fluorescent labelling as well as biotin-labelling techniques) from fresh/frozen and parallel FFPE samples.
12:20 Lunch on Your Own (Sponsored Technology Workshops Available)
2:00 Chairperson's Remarks
Improving Data Reliability
2:05 RNAprotect Saliva - The Perfect Stabilizer for Salivary RNA for Biomarker Discovery
Dr. David T. Wong, DMD, DMSc and Noh Jin Park, PhD, University of California Los Angeles, School of Dentistry, Dental Research Institute
Saliva is an emerging biofluid for molecular diagnostics. A saliva transcriptome signature (4 mRNAs) has been shown to be highly associative with oral cancer (ROC=0.95). In order to fully utilize the potential of the salivary transcriptome for disease diagnostics, we explore various RNA stabilizers: RNAlater, Superase and RNAprotect Saliva. We found that RNAprotect Saliva (proprietary Qiagen) is by far the perfect preservant for salivary RNA providing instant halting of RNA degradation (up to 3 months tested); room temperature compatibility; enhanced RNA recovery as well as enhanced complexity of RNA recovery from saliva.
| 2:35 RNA Quality - More than Just a Ratio
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Sponsored
by |
Dr. Rick
Conrad, Senior Scientist, R&D, Ambion Inc
Even today, RNA quality has been determined by the ratios of the OD260:280 and the 28S:18S
rRNA.Typically, the accepted values for these have been greater than 2 and 2, respectively. However, at
Ambion, we find that these ratios are very dependent on the method of tissue procurement and the tissue type.
Additionally, to generate the highest quality of RNA, we also control for DNA and nuclease contamination. Finally, we maintain the strictest standards of ethical tissue procurement, process validation and post packaging control. We will discuss each of the quality criteria in more detail. |
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3:05 Title to be Determined
Dr. Karol Thompson, FDA (invited)
3:35 Refreshment Break, Last Chance for Poster and Exhibit Viewing
FEATURED PRESENTATION
4:15 Chairperson's Remarks
4:20 Structural Variation in the Human Genome: New Insights for Diagnostics and Disease Study
Dr. Stephen Scherer, Senior Scientist, Genetics and Genomic Biology; Director, The Centre for Applied Genomics; Associate Chief, Research Institute, Hospital for Sick Children and University of Toronto
The first wave of information from the analysis of the human genome revealed SNPs to be the major source of genetic and phenotypic human variation. However, the advent of genome scanning technologies has now uncovered an unexpectedly large extent of 'structural variation' in the human genome. Rapidly accumulating evidence indicates that structural variants can comprise millions of nucleotides of heterogeneity within every genome, and are likely to make an important contribution to human diversity and disease susceptibility.
4:55 Panel Discussion: Considerations for Moving into the Clinic
5:30 Close of Conference
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