Thursday, April 12
Proteomic and Genomic Sample Preparation

8:30 Chairperson’s Opening Remarks

10:00 Grand Opening Refreshment Break in Exhibit Hall

11:00 Chairperson’s Remarks

11:05 Gene Expression Profiling from Difficult Samples such as FFPE
Gianfranco de Feo, Ph.D., Senior Director, Customer Solutions, NuGEN Technologies
Access to biologically and clinically relevant samples for the discovery, refinement, and validation of gene expression signatures reflecting clinical phenotypes has been hindered by the inability to generate high quality gene expression results from the most readily available tissue sources, formalin fixed, paraffin embedded tissues. NuGEN technologies has recently developed a linear, isothermal, and robust amplification approach to perform whole transcript amplification from very small amounts of total RNA (below 5ng input). 

This technology will not only allow researchers to perform splice variant analysis, but will also allow for the use of degraded RNA samples, such as RNA isolated from FFPE sources, in expression experiments. Data demonstrating the performance of the technology, including sensitivity, linearity, dynamic range, and differential expression accuracy will be shown. In addition, data demonstrating the performance of the approach to clinically relevant samples such as RNA isolated from FFPE tissue sources will also be shown.

11:35 Measurement of Gene Expression from Fixed Tissue: qNPA Validation of Biomarkers for Diffuse Large-B-Cell Lymphoma
Bruce Seligmann, Ph.D., Chairman & CSO, HTG, Inc.
Measurement of gene expression from fixed tissue using the lysis only qNPA multiplexed ArrayPlate assay measures the total RNA in the tissue including the “in situ” cross-linked mRNA. It is sensitive (using only 1/4th of a tissue slice), gives equivalent quantitative results as measurement from fresh or frozen tissue, and gene expression levels correlate with their protein product biomarkers measured in situ by immunohistochemistry. Three independent studies using snap frozen tissue had identified non-overlapping sets of biomarker genes, for a total of 36 putative biomarkers. All 36 were measured plus cell lineage genes. The validation of a biomarker set and correlation to therapeutic response and survival outcome will be reported.

These results demonstrate that archives of fixed tissue can now be easily mined for biomarker and target validation. Safety studies, clinical development, and diagnostic assays can be performed using fixed tissue without changing current tissue storage practice, to get gene expression results that correlate to protein biomarkers without any pre-qNPA sample prep.

12:45 Lunch on your own (Luncheon Seminar Sponsorship Available) 

3:30 Refreshment Break in Exhibit Hall

4:15 The Investigation and Use of Microheterogeneity in Human Plasma Proteins
Randall W. Nelson, Ph.D., The Biodesign Institute, Arizona State University
This presentation will focus on the investigation of microheterogeneity -- e.g., point mutations, slice variants or post-translational modifications -- occurring in human plasma proteins. Using mass spectrometric immunoassay, microheterogeneity is observed, characterized and cataloged in moderate-sized cohorts (100’s - to - 1,000’s) of healthy individuals. Microheterogeneity in diseased cohorts is characterized in the same manner, and molecular variants considered indicative of disease are subsequently used in the systematic development of multiplexed mass spectrometric immunoassays able to detect disease with high accuracy. Examples will be given for the investigation of cardiovascular disease.

4:45 Optimizing the Detection of DNA Based Biomarkers in Blood Plasma
Theo deVos, Ph.D., Vice President, Diagnostic Development, Senior Scientist, Epigenomics Inc.
Measurement of genetic and/or epigenetic markers in DNA from plasma or serum has great potential for the development of screening tests in oncology. To realize this potential, sample collection and processing must be optimized to measure low levels of a target biomarker in a background of normal DNA. We present our efforts to optimize collection and storage of plasma samples, as well as our workflow for sample processing and analysis of DNA based biomarkers. As a case study, we describe our experience validating Septin 9 DNA methylation as a plasma biomarker for colorectal cancer.

5:15 Molecular Profiling of Circulating Tumor Cells – RNA Preparation, Microarray Protocols and Fish Analysis
Mark Connelly, Ph.D., Vice President, Department of Reagent Development, Immunicon
Circulating tumor cells (CTCs) can be detected in blood from patients with metastatic and primary carcinomas. Over the past several years, the development of CellTracks® technology based on immunomagnetic capture and fluorescent characterization of CTCs has enabled accurate enumeration of CTCs at extremely low frequencies, on the order of 1 CTC per 7.5 ml of whole blood. Pivotal clinical trials have shown that the number of CTCs before treatment is an independent predictor of progression-free and overall survival in patients with metastatic breast cancer. Interim analyses of data from clinical trials in prostate and colorectal cancer appear to mirror data from the breast cancer trial. The talk will focus on antisense RNA library construction from CTCs obtained from hormone-refractory prostate cancer patients, as well as global gene expression profiling of CTCs and Circulating Endothelial Cells. FISH analysis and protocols will be also discussed.

5:45 Networking Reception in Exhibit Hall

7:00 End of Day Two

Friday, April 13
Genomic Sample Preparation

7:30 Coffee (Breakfast Technology Workshop Sponsorship Available)

8:30 Chairperson’s Opening Remarks

10:00 Refreshment Break in Exhibit Hall

10:45 Strategies for Validating Transcript Data in Biopharmaceutical Development
Eric R. Fedyk, Ph.D., Senior Scientist II, Drug Safety Evaluation, Millennium Pharmaceuticals, Inc. 
Valuable targets and biomarkers identified in discovery efforts often fail to impact downstream clinical programs. A number of variables can prevent translation of signatures from identification and validation, to utilization, some of which are controllable, whereas others are uncontrollable. A critical activity for successful utilization is to identify these variables, control them as best possible, annotate those that are uncontrollable, and analyze data accordingly.

11:45 Preparing and Handling Templates for High-Throughput Functional Genomics: Approaches and Challenges
Manish Biyani, Ph.D., REDS Group (JST), Rational Evolutionary Design of Advanced Biomolecules, Saitama Small Enterprise Promotion Corp., Japan
Development of advanced protein microarray is at the leading edge of functional genomics. Compared with DNA templates, proteins are much more difficult to produce and to handle. On the other hand, the large-scale genome sequencing projects have already been reveled that the complexity of proteome far exceeds that of the genome. Therefore, it becomes necessarily important to develop large-scale protein functional chip which can obviate protein handling problems and further can link it with its genotype. We addressed this challenge by developing a novel microarray platform for next-generation protein biochip which uses a novel solid-phase RNA-protein fusion technology [1] to create high-throughput functional protein arrays directly from encoding mRNA, such that the encoded proteins are immobilized on a surface as they are synthesized. This enables parallel analyses of high-density protein microarray where protein is insured to adhere onto the surface while properly retained not only their correctly folded functional content but also the genetic (encoding DNA/RNA) information content. Proof-for-concept validation of described scheme, i.e. “DNA-to-Protein chip”, will be presented and discussed.

12:15 Lunch on your own 
(“Lunch and Learn” Tutorial Sponsorship Available)

1:35 Excavating the Archive: Tools for Enabling Nucleic Acid Discoveries from FFPE Tissues  Rick Conrad, Ph.D., Senior Scientist, R&D, Tim Barta, Emily Zeringer, Ambion, A Business Division of Applied Biosystems The capability to isolate nucleic acid suitable for molecular analysis from formalin-fixed paraffin-embedded (FFPE) archived tissue samples enables the retrospective studies of a huge library of tissue representing various diseases, often through their progression. This data could be mined at both the genomic and gene expression level. While standard preservation techniques that employ formaldehyde are ideal for maintaining tissue structure and preventing putrefaction, this type of preservation usually interferes with molecular analyses on samples due to extensive chemical modification and subsequent fragmentation of the nucleic acids. Here, we report methods to facilitate genotyping, gene expression, and miRNA analysis from FFPE samples. The following practical elements of optimized FFPE workflows will be discussed: comparison of nucleic acid isolation protocols, accurate quantitation of functional template, strategies for enhanced genotyping confidence, selection of appropriate assays and amplicon sizes, and maximizing data retrieval from limited sample inputs.

Sponsored by:

2:05 Automated Target Preparation for Gene Expression Microarrays
Frederic Raymond, MSc, Microarray Lab Manager, Functional Genomics, Nestlé Research Center, Switzerland
While technical variability in sample preparation and chip processing may influence microarray-derived gene expression results, highly standardized and automated procedures can generate reproducible and comparable results across experimental replicates, studies, and even platforms and laboratories. We have therefore equipped our laboratory with a liquid handling system and programmed it to automatically perform all steps of the microarray target preparation, i.e. all procedures upstream the on-chip hybridization. Our fully integrated robotic platform can process up to 24 samples in parallel and reproducibly produces high-quality biotin-labeled cRNAs ready to be hybridized on one-colour oligonucleotide microarrays. We present technology development and applications.

2:35 Simulataneous Stabilization of RNA, DNA and Proteins in Tissue Samples
Vera Holländer, Ph.D., Senior Scientist, R&D, Qiagen GmbH
With gene expression profiling expanding to a systems biology approach, an increasing number of labs have started to analyze multiple analytes ­ DNA, RNA, and proteins ­ from the same sample. At the same time, multiple centres and repositories around the world have started to collect clinically relevant human sample material to be assessed by the research community in years to come. These future studies will potentially analyze yet unidentified targets with novel assay and detection technologies. Such undertakings rely critically on the conservation of the total molecular content of a sample. To support this growing need, we have developed a new technology that allows the simultaneous stabilization of RNA, DNA, and proteins in tissue samples. We have also established corresponding sample preparation technologies for the simultaneous purification of separate fractions of RNA, DNA, and proteins from a single sample.

3:05 Refreshment Break in Exhibit Hall

3:35 Lab on a Chip for DNA Diagnostics
Uppili Raghavan, CEO, Biomems, Simems Pte Ltd., Singapore
The lab on a chip (LOC) concept is to realize the functions of bio-laboratory in a silicon chip. The miniaturized biolaboratories are fabricated by photolithographic process developed in the microelectronics industry to form circuits, chambers, valves and channels in quartz or silicon substrate. Fluidic samples can be manipulated by placing valves, pumps in the chip and fluid can be diluted, mixed with other reagents or separated by another process on the same chip. In this paper, a microfluidic chip for DNA extraction and amplification is described. Silicon substrate is used to form microfluidic components in the chip.

4:05 How Microtechnologies Could Enhance Sample Preparation Solutions
Clemence Labat, Ph.D., Market Analyst, Micro & Nanotechnologies for Life Sciences and Chemistry, Yole Développement, France
Sample preparation remains a major bottleneck in laboratory workflow both for genomic and proteomicapplications. Today, sample preparation steps represent up to 60% of the analysis cost. Sample preparation steps are not only reagent and time consuming, but their quality and reproducibility are key to ensure analysis with a high quality and a high sensitivity. Microtechnologies have already provided high added value solutions for detection automation. We will present how microtechnologies and microfluidics are bound to fasten and automate the sample preparation process, also making it possible to integrate those different steps in one device. Case studies of development and commercialized products will be presented. 

4:35 Panel Discussion: The QC of Templates – Technology Users versus Providers

5:10 End of Conference