Sunday, October 22

5:30-6:30 pm Early Registration

Monday, October 23

8:00 am Registration and Morning Coffee

8:55 Chair’s Opening Remarks

9:30 Normalization of Gene Expression using Expressed Alu Repeat Elements
Jo Vandesompele, Ph.D., Center for Medical Genetics, Ghent University Hospital
The current golden standard for qPCR based gene expression normalization is the use of multiple carefully selected and validated reference genes. Many algorithms and accompanying software tools have been developed to select stably expressed reference genes from a panel of tested candidate reference genes (including our own geNorm platform). While all these methods work well in identifying proper reference genes, they require relatively extensive experimental work to do so. To address this issue, we have established an alternative for the reference gene concept. With the knowledge that some human genes contain one or more repeat sequences, we performed an in silico analysis of the human transcriptome. This indicated that a few thousand Alu repeat elements belonging to more than 30 families are expressed in as many as a few thousand different genes. By designing primer pairs that selectively amplify different Alu repeat families, we are thus simultaneously measuring the expression of many different transcripts, acting as an estimation of the general mRNA fraction abundance. Data will be presented on how expressed Alu repeat elements can be applied in accurate, experimental-validation-free normalization of primate gene expression.

10:00 Validation by Participation
Pamela Scott Adams, BS, Director/Manager, Molecular Biology Core Facility, Trudeau Institute
The major goal of the Association of Biomolecular Resource Facilities (ABRF) Research Groups is to define and improve the technical capabilities of scientific laboratories by providing educational material and benchmark tests that enable participants to gain positive feedback on their technique, reagents and instrumentation in an anonymous fashion. Research group studies are run annually covering a diverse gamut of DNA (and protein) technologies, including Microarray and Real-time PCR. Recent studies by the Nucleic Acids Research Group (NARG) on quantitative PCR have included surveys to monitor qPCR practices, and studies on Taqman® probe synthesis, primer design, standard curve performance and the effects of reverse transcription on real-time PCR results. Participation in these benchmarking studies can be an important tool for technique validation.

10:45 The 'Gold Standard' of Gene Expression Measurements is an Elusive Goal
Timothy J. Triche, M.D., Ph.D., Chief Pathologist, Department of Pathology & Laboratory Medicine, USC Keck School of Medicine Childrens Hospital Los Angeles
Many authors have addressed the lack of uniformity in gene expression measurements using microarray technology. Alternative measures such as quantitative PCR have been proposed as a 'gold standard' to amend these perceived deficiencies. However, this 'solution' has its own deficiencies that suggest it is not an independent 'gold standard' in the absence of corroborating biological knowledge. As an example, PCR primers that span two or more exons will not account for alternate splicing, which is widespread among human genes. Further, PCR values are typically not absolute, but relative, and thus compared to what reference point? Use of protein measurement methods is likewise no absolute index, as it is well known that there is no 1:1 correlation between mRNA transcript levels and protein synthesis and accumulation. Finally, much of the variance in reported expression values from microarray analysis can be corrected with suitable data pre-processing algorithms, each of which makes its own assumptions about the data. Thus, there is no simple 'gold standard', and results from any of these methods requires some degree of sophistication in analysis and interpretation. Examples of each will be presented and discussed.

12:00 Lunch on Your Own
(Technology Workshop Sponsorship Available)

2:05 Microarrays & QPCR: The Dynamic Duo of Validation
Lisa White, Ph.D., Director, Microarray Core Facility, Baylor College of Medicine
Cross-platform validation is an important component of expression profile analysis. Data obtained from microarray-based platforms can be validated using multiple methods—alternative microarray platforms, quantitative RT-PCR (QPCR), northern blot, and in situ hybridization. We utilize microarray and QPCR data together as validation tools as well as complementary platforms to provide the most extensive coverage and greatest confidence for researchers asking complex biological questions. I will discuss the integration of microarray and QPCR platforms in our core facility and its utility in providing more complete answers to these questions. Case studies will be presented that cover experimental design issues, using QPCR to validate microarray data, post-microarray design of QPCR panels to validate large sample sets, and use of QPCR for more comprehensive pathway analysis.

2:35  From Brain to Blood: Discovery, Validation and Development of a SNP Assay for Detection of Increased Risk of Suicide in Major Depression using Gene Expression Microarrays
Eric Kaldjian, Ph.D., Senior Scientific Director, GeneLogic Inc.
This presentation will discuss the use of gene expression microarrays to identify differentially regulated transcripts in brain regions of patients with major depression and depression combined with suicide. Gene expression was investigated in the regions BA 4, 8/9 and 11. Decreased expression relative to controls of one of the genes, spermine/spermidine N-acetyltransferase (SSAT), was validated by RT-PCR, western blot and immunohistochemistry. A genetic variant (SSAT342A/C) was found to have a statistically significantly association with gene expression level (P = 0.02). A SNP assay for the variant was developed and applied to blood samples in an independent population of male suicide completers and controls. The SSAT342C allele was found to be present in higher frequency among suicide cases (P = 0.005), suggestive of an increased predisposition to suicide in individuals with this allele. This case example demonstrates a process for technological and initial clinical validation of a biomarker derived in diseased tissue and assayed in the blood. 

3:05 Whole Blood Gene Expression using Microarrays and StaRT-PCR
Shelley Ann des Etages, Ph.D., Senior Principal Scientist, Genetic Technologies, Pfizer, Inc.
Transcriptomics of target tissues is widely applied in monitoring drug response in the pre-clinical setting. In a clinical setting, access to target tissue may be limited or absent and as such it is important to understand whether whole blood profiling can be used to draw comparable conclusions. Genome-wide and targeted gene expression completed on human whole blood samples collected in a controlled clinical trial demonstrated the consistency of gene expression in whole blood and the normal range of expression levels for a subset of transcripts.

3:35 Gene Silencing Analysis: Complimentary use of RT-qPCR, 2-DGE, and Western blotting for RNAi-based pathway analysis
Hilary K. Srere, Ph.D., Marketing Manager - Amplification, Gene Expression Division, Bio-Rad Laboratories 
RNA interference (RNAi) is a powerful tool used to modulate gene expression and to determine gene function. In this study, we employed a multifaceted approach to examine changes of protein and gene expression profiles in HeLa cells after knocking down the cytoskeleton protein, â-actin, using 27-mer siRNAs. 

3:50 Technology Spotlights 

4:05 Refreshment Break, Poster and Exhibit Viewing

5:30 Networking Reception

6:30 Close of Day One