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Tissues, Proteins, DNA or RNA - to ensure the production of reliable, high quality data, it is critical to set high standards at the beginning, since the results depend greatly on the quality of original samples. Despite improved technologies, many issues still need to be discussed. What are the best ways to store Proteins, DNA and RNA and what are solutions for long term storage? How can reproducibility be improved? Can there be a consensus on "standardization?" Upstream sample
preparation - what are the current bottlenecks? Key scientists from academia, industry, and government are coming together for exciting discussions, presentations, and collaborations around the most current issues and solutions in the world of sample preparation. Come bring your knowledge and questions and join them!
4:00-5:00pm Early Conference Registration
Monday, April 24
7:30-8:30am Registration
and Morning Coffee
Opening Plenary Session
| KEYNOTE PRESENTATIONS
8:30 Chairperson’s Opening Remarks
Dr. James L. Wittliff, Professor of Biochemistry & Molecular Biology, James Graham Brown Cancer Center, University of Louisville
8:40 Challenges in the Preparation of Clinical Samples for Genomic and Proteomic Analysis
Dr. Scott D. Patterson, Senior Director, Medical Sciences, Amgen Inc.
Obtaining samples from clinical trials for subsequent genomic and
proteomic-based studies can be challenging. Assays that perform well in the research laboratory setting can pose problems when translated to the clinical setting. It may be that a portion of the assay has to be conducted at the clinical site and the remainder in your own laboratory. This presentation will discuss some examples of such assays and how they were conducted successfully.
9:20 Effective BioBank Management in an Age of Uncertainty
Dr. Richard Hegele, Professor and Head, Department of Pathology and Laboratory Medicine, James Hogg iCAPTURE
Centre, University of British Columbia, Canada
Biobanks and other types of biological repositories are a tremendous resource not only for current research but for future research in areas we cannot even begin to imagine at present. At the management, operations and regulatory levels, this presents a number of challenges for continued use of previously obtained specimens, in light of new privacy legislation and other developments. This presentation will review how the James Hogg iCAPTURE Centre has addressed these challenges, by harmonizing its separate cardiovascular and pulmonary tissue registries, its consent processes in light of new legislation, and activities in quality
assurance. |
| 10:00 Morning Coffee |
Sponsored by
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Sample Partitioning - Fractionation
10:30 Chairperson’s Remarks
Dr. Jerry Feitelson, Manager of Strategic Marketing, Proteomics Business Center, Beckman Coulter
10:35 Plasma Proteomic Sample Processing: Development of a Robust Workflow Intended for Large Scale Studies
Dr. Moira Lynch, Scientist, BG Medicine
Proteomic studies of disease and drug effects typically require the quantitative analysis of hundreds of samples. Optimizing the value of these analyses requires striking a balance between protein coverage, throughput, and reproducibility/ robustness. In order to be able to run large scale proteomics studies (100-1000+ samples) it was our challenge to show that a multi-step plasma proteomics workflow could be performed uniformly for all of the samples in a study. The first phase was to develop and optimize each step in the workflow and implement QC methods to monitor performance and consistency. A mixed-bed IgY column was used to remove twelve abundant proteins in plasma by affinity chromatography. Then reduced and alkylated proteins were digested and the peptides were labeled with iTRAQ Reagents allowing for multiplexing of samples and relative quantitation through MS/MS analysis. Four samples were pooled after iTRAQ labeling and this single-tube processing approach minimizes the impact of any process-to-process variation occurring in subsequent steps. The iTRAQ
four-plexes were analyzed through SCX-HPLC MS/MS using both MALDI and electrospray ionization. Exclusion and inclusion lists were used extensively in LC-MS/MS to maximize the consistency of peptide sets measured throughout the study. Once the workflow was robust, there were other considerations impacting the design of large-scale studies. In order to be able to compare any pairs of samples in the study, a reference pool was created that provided a fixed member in each
four-plex iTRAQ-labeled sample mix. To minimize the effect of temporal fluctuations in the efficiency of analytical steps, aliquots of the reference sample were processed together with those triplets designated for the same
four-plex mix. Additionally, specific sample randomization schemes were utilized to optimally segregate the effect of biological variations from random analytical variations. These large scale studies are now routinely run in our laboratory due to all these considerations.
11:05 Divide to Conquer: Proteomic Fractionation of Plasma Samples for Low Abundant Biomarker Discovery
Dr. Sunny W. Tam, Professor, UMMS Proteomic Consortium, University of Massachusetts Medical School
State of art immunoaffinity depletion and liquid IEF techniques have been used to partition the highly abundant plasma proteins from the lower abundant proteins. Subsequent high-throughput zoom 2D gels, in conjunction with a panel of highly sensitive dyes for
post-translational modifications and quantitation, allows the detection of known and novel biomarkers in plasma. The presentation will demonstrate adequate upfront fractionation of plasma samples as the key to simplify the complexity of plasma proteome and allow detection of low abundant proteins for subsequent 2D gel or MS analyses.
11:35 Affinity-Enrichment Mass Spectrometry with Mass Defect Tags for Biomarker Validation
Dr. Luke V. Schneider, Chief Scientific Officer, Target Discovery, Inc.
Current global proteomic approaches often fail to penetrate deep enough into the proteome to identify known low abundance biomarkers because of the limited dynamic range of mass spectrometry. Global approaches can also be adversely affected by variations in sample collection and storage between clinical sites and are far too expensive for biomarker validation, which involves screening 100’s to 1000’s of patient samples, or routine clinical use. Mass defect tagging allows for the affinity enrichment of low abundance protein biomarkers out of
biofluids, such as serum, prior to mass spectrometric analysis. Mass defect technology increases throughput and recovery since further simplification of the sample (e.g., ion exchange and reverse-phase high performance liquid chromatography) is not required because the 0.1 amu shift imparted by the mass defect tag allows the target peptides to be readily discriminated from the antibody or bait proteins. When coupled with stable isotope paired reagents, mass defect tags allow high precision quantification of biomarker levels and/or internal standards to be used directly in clinical samples. This technology is perfectly suited for validation of putative biomarkers with large sample sets with sub- 10% quantitative precision. Cysteine tags can be used to measure up and down regulation of more than 200 biomarkers simultaneously in multiplexed assays. Lysine tags allow protein isoform differences to be analyzed because they provide greater sequence coverage than cysteine tags. We illustrate the use of IDBEST™ with the enrichment of prostate specific antigen
(PSA) and alpha-fetoprotein (AFP) from human serum.
12:05pm Lunch on Your Own
Downstream Sample Prep
2:00 Chairperson's Remarks
Dr. Alexander Lazarev, Director of Reasearch and Development, Pressure BioSciences, Inc.
2:05 Proteomic Sample Preparation for Studying Animal Host Response to Pathogens by 2-D DIGE
Dr. Brett Chromy, Biomedical Scientist, Defense Biology, Lawrence Livermore National Laboratory
The projects we are currently working on are solving some of the problems with studying animal
proteomes, including the use of 2-D DIGE for improved detection of differentially expressed proteins and depletion of high-abundant proteins to study the more relevant putative biomarkers from a variety of animals and their tissues. We study host response to pathogens, such as Yersinia
pestis, Bacillus anthracis, and Salmonella enterica serotype Typhimurium. We also have developed a plasma proteome sample preparation for studying a pig model of staphylococcal enterotoxin B
(SEB) intoxication. Our work represents the first study of Y. pestis response in mice using 2-D DIGE examining multiple tissue samples. Our work can be of interest to researchers looking for improved proteome preparations from diverse animal models, as well as those studying host-pathogen interactions.
| 2:35 Panning for Biomarker Gold: Applying Antibody-Mediated Proteome Partitioning and 2D Fractionation to Biomarker Discovery |
Sponsored by |
Dr. Jerry Feitelson, Proteomics Business Center, Beckman Coulter
A proteome may be a treasure trove for biomarker discovery. However, it is clear that such complexity and breadth of dynamic range in protein concentration create a significant challenge for today’s analytical technologies. As a result, strategies for simplifying the sample prior to analysis are being developed. In this presentation, we propose a partitioning & fractionation strategy that utilizes selective enrichment techniques such as immunoaffinity partitioning upstream of multi-dimensional liquid fractionation. We will discuss enrichment techniques for low abundant proteins, methods to alleviate the masking effect of protein mass fingerprints from high abundance proteins, as well as automated two dimensional proteome fractionation by isoelectric point and
hydrophobicity. Specific examples from the proteomics research community will be presented. |
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3:05 Proteome-Wide Analysis of Formalin-Fixed Paraffin Embedded Head and Neck Cancer Tissues Using Laser Capture Microdissection and Mass Spectrometry
Dr. Vyomesh Patel, Staff Scientist, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH
We have conducted the first proteome-wide analysis of Head and Neck Squamous Cell Carcinoma
(HNSCC) by mass spec proteomic profiling of formalin fixed archival HNSCC tissue. In this study we have combined laser capture microdissection (LCM) and Liquid TissueTM protein extraction reagents with mass spectrometry to identify proteins expressed in normal and cancerous oral squamous epithelium. Up to 900 proteins per sample were identified using strict statistical criteria, some of which correlated with tumor progression. Our data indicate that the combination of LCM and large scale proteomic analysis of fixed archival cancer tissue may represent a suitable approach to investigate complex changes at the protein level in cancer. These efforts may likely result in the identification of new biomarkers for HNSCC that can be used to diagnose disease, predict susceptibility, and monitor progression in individual patients.
3:35 Refreshment Break, Poster and Exhibit Viewing
4:15 Give-and-Take Roundtables
Give-and-Take Roundtables are one hour focus groups designed to discuss important and challenging topics related to Proteomic Sample Preparation, Tissue Technologies and Labels and Stains for Tissue and Cells. This is a moderated discussion with brainstorming and interactive problem solving between scientists from diverse areas who share a common interest in the discussion topic.
These forums are for open discussion of scientific challenges, and not sales opportunities. We emphasize that this roundtable is for an interactive exchange among scientists and is not meant to be, in any way, a corporate or product discussion.
5:15 Networking Reception in Exhibit Hall
6:15 Close of Day One
Tuesday, April 25
7:30am Morning Coffee, Breakfast (Technology Workshops Sponsorship Available)
Opening Plenary Session
| KEYNOTE PRESENTATIONS
8:30 Chairperson’s Opening Remarks
8:40 Triage for Predicting Clinical Outcome using Molecular Profiling of Human Carcinoma Cells Procured by LCM
Dr. James L. Wittliff, Professor of Biochemistry & Molecular Biology, James Graham Brown Cancer Center, University of Louisville
Forecasting clinical behavior and therapeutic response of human cancer currently utilizes a few tumor markers and disease characteristics. Medically relevant genomic and proteomic test development requires a troika consisting of human tissue specimens collected and stored under specialized conditions with annotated clinical records, isolation of distinct cell types with technologies such as laser capture microscopy and analyses of macromolecules with protease and
RNase-free protocols to generate gene and protein expression profiles. Using an extensive Biorepository of frozen and
formalin-fixed, paraffin-embedded tissue specimens with long-term clinical follow-up, pure populations of cells were procured by LCM for RNA extraction, amplification and
microarray. Zinc-finger binding proteins, analyzed as models for
proteomics, were identified using 32P-labeled hormone response elements and band-shift assays. Genes associated with estrogen receptor-alpha (ER) status dominated expression profiles suggesting a larger hormone-related network than previously appreciated. Other prominent gene expression patterns divided ER+ carcinomas into two subgroups, each associated with significantly different clinical outcomes. Another gene expression signature was identified strongly correlated with breast cancer recurrence using supervised learning methods. This large retrospective investigation of pure carcinoma cell populations identified molecular signatures highly associated with clinical outcome suggesting promise for development of novel prognostic tests for breast cancer management and identification of new molecular targets for drug design.
9:20 Cells, Microchips, and Clinical and Biological Information
Dr. Mehmet Toner, Professor of Biomedical Engineering, Harvard Medical School, Massachusetts General Hospital
Microfabrication techniques that have revolutionized the electronics industry, are now poised to revolutionize the pharmaceutical, biotechnology, and biomedical device industries. The two leading applications of microfabrication in biology include “genes-on-a-chip” to monitor the expression level of potentially all genes in humans or various model systems and organisms simultaneously, and “lab-on-a-chip” type devices to perform biochemistry in
microchambers. Equally exciting is the fact that biomedical application of microfabricated devices is no longer limited to non-living systems such as genes-on-a-chip or lab-on-a-chip. Recent advances in the understanding of cellular behavior in microenvironments have started to pave the way towards living micro-devices. The ability to integrate cells with microdevices is important for controlling cellular interactions on a sub-cellular level, for obtaining highly parallel, statistically meaningful readouts over large cell populations, and for miniaturizing instrumentation towards minimally-invasive, portable, fast, inexpensive devices. The rich assortment of living microdevices that can be built using BioMEMS techniques have important applications in fundamental cellbiological studies, tissue engineering, cell separation and culture devices, diagnostics, and high-throughput drug screening tools. This presentation will provide an overview of some of the key advances and remaining challenges related to the combination of microfabrication technology and living cells and a sampling of a broad spectrum of exciting opportunities and promises in biology and medicine. |
10:00 Morning Coffee, Poster and Exhibit Viewing
Setting Standards
10:45 Chairperson’s Remarks
Dr. Birgit Jostes, Senior Global Director, Corporate Marketing and Strategy, QIAGEN GmbH
10:50 Productivity and Financial Implications Associated with a Lack of Standardization in Proteomics Sample Prep
Mr. Bruce Haywood, Business Development Leader, Clinical Proteomics, BD
It is becoming crystal clear that “business” issues are stultifying progress in clinical
proteomics, as much as the scientific challenges. However, a clear delineation of these issues, and their stark implications, has yet to be cohesively described in a suitable manner for broad public consumption. We can significantly accelerate progress through more profound, smarter long-term collaborations and efforts that vector toward standardization and compliance.
11:20 Pressure Cycling Technology: A New Paradigm in Sample Preparation for Genomics, Proteomics, and Metabolomics
Dr. Alexander Lazarev, Director of Reasearch and Development, Pressure BioSciences, Inc.
Recent advancements in the quantitative analysis of nucleic acids, proteins, and small molecules clearly illustrate that sample preparation can introduce significant analytical variability, while concomitantly engendering major bottlenecks to the workflow. This presentation will focus on Pressure Cycling Technology (PCT), a novel method used to disrupt tissues and cells and to facilitate solubilization of wide variety of analytes. Cell suspensions or tissues, are subjected to alternating cycles of high (up to 35,000 PSI) and ambient pressure, precisely controlled by specialized instrument called Barocycler. PCT exploits physical properties of the biological material, which enables efficient sample homogenization in simplified reagent systems that are compatible with downstream analytical processes, such as PCR, hybridization, 2D gel electrophoresis, and mass spectrometry. Because samples are enclosed in single-use inert containers, the possibility of cross-contamination is significantly reduced, while the level of occupational safety for researchers when infectious or toxic materials are processed is increased. In this presentation, advantages of the PCT Sample Preparation System will be illustrated in comparison with conventional methods such as ultrasonic cell disruption, Polytron shearing, and grinding with mortar and pestle. Applications of PCT in genomics, proteomics, and adjacent disciplines will be demonstrated using a variety of cell and tissue samples and downstream analytical techniques.
11:50 Reproducible Profiling and Biomarker Development in Whole Blood Assays
Dr. Chris B. Russell, Molecular Sciences, Amgen, Inc.
| 12:20pm Luncheon Seminar |
Sponsored by |
Achieving Standardisation of Pre-Analytical Workflows through Automation
Dr. Anja Schultz, Senior Scientist, R&D, QIAGEN GmbH
During recent years researchers in the field of molecular biology have turned from studying individual aspects such as DNA sequence, RNA expression, or protein structure to more comprehensive, system biology type approaches. Results are being entered into public data bases and are discussed in worldwide networks of research groups. This requires highly standardised and controlled processes to generate comparable and consistent data. Automation of pre-analytical and analytical workflows can be a means to eliminate human influence and to help standardising analyses between different labs. We have designed instrument solutions for automating pre-analytical workflows with the goal to maximize standardisation and ease-of-use. Two automation solutions, the BioRobot EZ1, and the BioRobot Universal System will be described, both carrying a broad range of protocols for RNA, DNA, and protein purification from a variety of starting materials. |
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Preparing Genomic Samples
2:00 Chairperson’s Remarks
2:05 Quantitative Gene Expression Results from Fixed Tissue Without Specialized Sample Preparation
Dr. Ralph Martel, VP Research and Development, Management, HTG
The measurement of gene expression from fixed tissue has been at best problematic, time consuming, and cumbersome. However, using gene expression assay technology, fixed tissue can be dissolved in lysis reagent and tested using the same simple protocol that is used for cells or frozen tissues, and it provides the same quantitative measurements of gene expression levels. Data from matched frozen and fixed tissue will be presented, including experiments where tissue fixed years earlier is compared to freshly fixed tissue from the matched frozen block. The multiplexed format of the technology permitting up to 16 genes to be measured in each well of a
microplate, and the throughput of both the qNPA sample processing and the measurement, means that extensive retrospective studies are now possible to validate gene signatures, targets for drug discovery, and biomarkers for diagnostic applications. The impact of this breakthrough on the target validation and drug discovery process will be discussed.
2:35 Nanoparticle-Based DNA Purification for PCR
Dr. Michael Hogan, Managing Director, Scientific Affairs, Argylla Technologies LLC
We describe here a simple, inexpensive and highly efficient form of batch chromatography, based on chemically
coated,sub-micron ceramic particles (nanoparticles) for the capture, purification and concentration of DNA for
PCR-based genomics. Because of their sub-micron size, coated ARGYLLA nanoparticles present an enormous surface area to volume ratio
(100cm2/uL) which generates a high DNA binding capacity (2mg DNA/uL). The nanoparticles are small enough that they form a stable suspension at 1g (where they can bind DNA effectively) but because they are dense, they are readily harvested by low speed centrifugation (2000g) to produce a discrete, white, microliter pellet, from which captured, purified DNA may be released at room temperature into a volume as small as 5uL: as a
PCR-ready solution. Applications are described where chemically coated Argylla nanoparticles are used to isolate DNA
forhigh-value applications: forensic database building (cheek wipes) crime scene analysis (surface wipes) population genetics (dried blood on FTA paper) and clinical genetics (fresh blood, paraffin-embedded thin sections). As assessed by quantitative
PCR, both DNA yield and quality are found to equal or exceed that obtained by the use of industry-standard spin columns or magnetic bead products
3:05 The Collection, Archiving and Isolation of DNA from Dried Clinical Samples for Molecular Analysis
Dr. Michael Harvey, Principal Scientist, R&D, Whatman
The collection and preparation of clinical samples for molecular analysis is important for expanding applications in drug discovery, predictive medicine and
pharmacogenomics. Whatman has developed a novel technology, FTA and FTA Elute, for dried clinical sample collection, archiving and, most importantly, DNA preparation for amplification. With this technology sample preparation from samples collected on treated samples is simple, rapid and
automatable. Blood and buccal cell samples collected on FTA® and FTA® Elute can be stored for long periods of time under ambient conditions. This presentation will review studies that will demonstrate the use of DNA from the aforementioned types of samples for forensic identification, real time
PCR, DNA sequencing and allele
specific hybridization methods.
3:35 Refreshment Break, Poster and Exhibit Viewing
Closing Plenary Session
| FEATURED PRESENTATIONS
4:15 Chairperson’s Opening Remarks
4:20 Molecular Maps of Prostate Cancer: Tissue Print Micropeel Profiles of Human Surgical and Biopsy Specimens
Dr. Sandra Gaston, Principal Investigator, Department of Surgery, Beth Israel Deaconess Medical Center
Recent advances in molecular technologies have led to the acceleration in the identification of potential biomarkers for human cancers. To realize the full potential of this wealth of new biomarkers in the development of targeted therapies for solid tumors, it is essential that we develop new strategies for profiling human tissue specimens that are compatible with the demands and restrictions of the clinical setting. My laboratory has developed a set of novel tissue print micropeel techniques that allow us to map a series of molecular marker profiles from an extended area of a human tissue sample without damaging the specimen. Because our tissue print platform supports both proteomic analysis and
PCR-based DNA and mRNA profiling techniques, it can be used to generate detailed molecular cross-sections that can be layered directly over corresponding histological and radiological images. Such extensively annotated maps of human tissue and tumor specimens can be used for target-based tumor classification and for monitoring molecular responses to target-based therapies. I propose, in my talk, to present an overview of our tissue print micropeel technologies, with specific examples of the application of these technologies in the molecular assessment of human tissue biopsy and surgical specimens.
4:55 RNA Quality and Yields from Frozen Tissues: Analysis of Samples from a Large International Repository
Dr. James Eliason, Chief Scientific Officer, Research and Development,
Asterand, Inc., Adjunct Associate Professor of Oncology and Medicine, Karmanos Cancer Institute, Wayne State University
Frozen tissue repositories are an important resource for genomics and proteomics research. When repositories contain samples from multiple sites, it is important to have ways to assess the quality of the samples. RNA is highly sensitive to handling and freezing conditions. We have routinely performed an RNA quality control test for every sample we have received. We have compared several different measures for RNA quality including the RNA Integrity Number proposed by
Agilent. We have also examined several factors that affect RNA quality and yields. |
5:30 End of Conference
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