Enabling Molecular Profiling with Cellular Resolution

Researchers are increasingly analyzing homogeneous cell samples to obtain greater specificity and sensitivity in molecular assays instead of larger tissues containing mixed cell populations. Laser capture microdissection, FACS sorting, and magnetic bead capture are among the methods developed to obtain cells of the same type from tissue or blood. But resulting samples are often quite limited, as is the level of specific molecules being measured. What are the challenges of doing genetic, gene expression, genomic, proteomic, and metabolomic analysis? How can they best be addressed? What can you learn from the experiences of researchers working with these samples in a variety of applications? Experts in the technologies and applications of molecular analysis at the resolution of pure cell populations are brought together at this conference. Data from these homogeneous populations are proving to be much more informative than that from mixed cell samples. Productive research is clearly headed in this direction. Come be a part of the discussions and presentations of results reflecting this shift.

Keynote Address
Cellular Resolution in Molecular Profiling
Dr. Lance Liotta, National Cancer Institute

Session Chairs
Dr. Mark G. Erlander, Arcturus
Dr. James F. Leary, University of Texas Medical Branch
Dr. G. Mike Makrigiorgos, Dana-Farber Cancer Institute and Harvard Medical School
Dr. Jean Rossier, CNRS

Additional Speakers
Dr. Marie Bosnes, Dynal Biotech ASA
Dr. Yanxiang Cao, Affymetrix Inc.
Dr. Anthony G. DiLella, Merck & Co., Inc.
Dr. Mabel Duyao, Ardais Corp.
Dr. Michael C. Ellis, Renovis, Inc.
Ms. Susan M. Goldsworthy, GlaxoSmithKline
Dr. Howard B. Gutstein, University of Texas-MD Anderson Cancer Center
Dr. Eric Henderson, BioForce Nanosciences, Inc.
Dr. Manfred R. Koller, Cyntellect, Inc.
Dr. Nurith Kurn, NuGEN Technologies, Inc.
Dr. G. Mike Makrigiorgos, Dana-Farber Cancer Institute and Harvard Medical School
Dr. Bernd Meurers, Johnson & Johnson Pharmaceutical Research and Development
Dr. Mats Nilsson, Uppsala University
Dr. Renee A. Reijo Pera, University of California, San Francisco
Dr. Daniel W. Rosenberg, University of Connecticut Health Center
Dr. Mark A. Watson, Washington University School of Medicine
Dr. Joanne M. Yeakley, Senior Scientist, Illumina, Incorporated

Sample Preparation
Getting the Right Cells to the Microarray
High-Resolution Molecular Analysis: Applications in Clinical Genomics
Combined Isolation of Pure Cells, Nucleic Acids, and Proteins
High-Purity, High-Yield Cell Isolation

Gene Expression Profiling: Technologies
Automated Gene Expression Profiling on Randomly Assembled Universal Arrays
Analysis of RNA from Laser Capture Microdissection Cells
Applying Microarray Gene Expression Analysis to Microscale Samples
Microdissection, Transcript Amplification, and High-Density
Oligonucleotide Microarray Analysis
Balanced PCR: New Method for Retaining the Difference between Two
PCR-Amplified Complex Genomes
Novel Isothermal Linear Amplification Method for mRNA

Gene Expression Profiling: Applications
Understanding Molecular Mechanisms Underlying Human Reproduction
through Global Gene Expression Profiling
Novel Molecular Signatures Discriminating Stage and Grade of Breast Cancer
PBK (PDZ-Binding Kinase) as a Tumor Marker
Gene Profiling of Precancerous Lesions in Mouse Colon Using Laser Capture Microscopy
Gene Expression Analyses in the Central Nervous System
Neurons That Matter: Profiling Specific Cell Types for Target Discovery

Proteomics and Metabolic Profiling
Transcriptome, Proteome, and Metabolome of a Single Cell: Patch Clamp Pipettes Harvesting
Targeted Proteomic Analyses of Small Cell Groups
Nanoarrays for Proteomics
Padlock- and Proximity-Probe Ligation Assays for Localized Detection of
Single Molecules and for Parallel Analysis of Genes, Transcripts, and Proteins

11:00- 12:00am Registration for Preconference Tutorial

12:00-5:00pm Preconference Hands-on Tutorial

This hands-on tutorial will introduce participants to Arcturus' Systems for Microgenomics integrated and complete tool sets for reproducible extraction and analysis of biomolecules from pure cell populations. This will include talks on tissue preparation, Laser Capture Microdissection (LCM) and how such samples can be used for DNA, RNA and protein analysis using Arcturus' platforms. Participants will be able to perform microdissections and acquire homogeneous cell populations using the PixCell® IIe Laser Capture Microdissection System and be able to speak with Arcturus scientists about downstream sample analysis. A detailed demonstration of the AutoPix® Laser Capture Microdissection (LCM) System, our latest innovation in the area of automated microdissection, will also be a part of this program.

7:30am Conference Registration, Poster and Exhibit Viewing, with Light Continental Breakfast

8:30 Chair's Opening Comments
Dr. James F. Leary, Assistant Vice President for Advanced Technology, University of Texas Medical Branch

8:40 Keynote AddressCellular Resolution in Molecular Profiling
Dr. Lance Liotta, Chief, Laboratory of Pathology, National Cancer Institute

9:20 Getting the Right Cells to the Microarray: The Importance of High-Throughput Cell Separation Technologies to Genomics and Proteomics
Dr. James F. Leary
The full promise of genomics has not yet been realized due to (1) gene expression contributions of other cell types in heterogeneous mixtures, (2) the need for new cell separation technologies and techniques for the purification of more homo-geneous cells from these heterogeneous cell mixtures and (3) limitations in cur-rent gene array technologies in dealing with small numbers of purified cells. In this presentation we show the effects of cell mixtures on gene expression profiles and multiple ways cells can be separated and purified prior to gene array analysis. We also discuss some of the problems, and some potential solutions, to the diffi-culties associated with amplification of mRNA from small numbers of cells - one of the core problems of the new field of "microgenomics".

9:50 High-Resolution Molecular Analysis: Applications in Clinical Genomics
Dr. Mabel Duyao, Ardais Corp.
The field of clinical genomics-the marriage of large-scale technologies for molecular analysis with the study of actual human disease-is rapidly becoming a key post-genomic strategy for increasing success in developing effective new therapeutics and diagnostics. Addressing challenges of an optimized clinical genomics approach, Ardais has worked with leading medical centers to develop the Biomaterials and Information for Genomic Research (BIGR™) Discovery Platform and a comprehensive set of bioethics policies and procedures, combining a proprietary information system, technical protocols, and logistics capabilities that streamline standardized and comprehensive collections of tissue and other clinical samples, along with highly structured associated clinical information. For higher resolution samples analysis, Ardais has also developed tissue microarrays in flexible formats for high-throughput in situ-based studies and implemented an optimized approach for the extraction of molecular derivatives from cell populations retrieved through Laser Capture Microdissection (LCM).

11:00 Combined Isolation of Pure Cells, Nucleic Acids, and Proteins from Single Samples
Dr. Marie Bosnes, Senior Research Scientist, Dynal Biotech ASA
The availability of pure cell populations is crucial for the analyses of gene expression, for both transcriptomic and proteomic profiling. Downstream analyses are becoming more and more automated, and sample preparation, perhaps the most important part of the study, is becoming the bottleneck. Dynal Biotech's magnetic bead-based technology is a simple, in vitro model system for isolating specific cells, genomic DNA, mRNA, and proteins with high documented purity. Specific cell isolation methods focus on minimizing the probability of affecting the physiological state (mRNA and protein content) of the cells, enabling downstream molecular characterization and profiling. Dynamic change of both mRNA and proteins result in profiles representing only snapshots of the cells' biology. Due to regulatory mechanisms at the transcriptional, translational, and post-translational levels, mRNA and protein levels do not always correlate, making it crucial to study both mRNA and proteins from the same sample. This technology is well suited for handling small samples, down to single cells. The generation of solid-phase cDNA libraries, which can be reused in multiple PCR analyses, will also be presented. All the methods are highly scalable and automatable.

11:30 High-Purity, High-Yield Cell Isolation for Gene Expression Analysis on the LEAP™ Technology Platform
Dr. Manfred R. Koller, Vice President, Research & Development, and Chief Operating Officer, Cyntellect, Inc.
A novel automated imaging and laser-based processing technology for the analysis and manipulation of individual cells in a high-throughput manner has been developed. The Laser-Enabled Analysis and Processing (LEAP™) platform has a number of unique attributes including a pulsed laser with galvanometer steering for rapidly manipulating individual cells and has been used to purify cells by firing the laser at unwanted cells, thereby eliminating them from the population. This allows the purification of cells on any transparent surface (including adherent cells in situ). In situ processing allows the high-throughput LEAP platform to easily process several cells to several million cells with high yield. The technology is currently being used to purify rare cell populations for gene expression analysis in an HIV infection model.

1:45 Chair's Comments
Dr. G. Mike Makrigiorgos, Department of Radiation Oncology, Dana-Farber Cancer Institute and Harvard Medical School

1:50 Automated Gene Expression Profiling on Randomly Assembled Universal Arrays
Dr. Joanne M. Yeakley, Senior Scientist, Illumina, Incorporated

2:20 Analysis of RNA from Laser Capture Microdissected Cells
Dr. Anthony G. DiLella, Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc.

2:50 Applying Microarray Gene Expression Analysis to Microscale Samples: Promises and Challenges
Dr. Yanxiang Cao, Program Manager, Gene Expression Research, Affymetrix Inc.
To meet the growing demand for quantitative gene expression analysis in microscale samples, Affymetrix has developed a highly sensitive amplification method for gene quantification measurements in 100 to 1,000 cells using GeneChip probe arrays. We have characterized the assay and applied it to obtain expression profiling of isolated specific neurons in the brain. The proof-of-principle study has demonstrated the feasibility of this approach and also revealed the promises and challenges in small-sample gene expression analysis.

4:00 Accurate and Reproducible Gene Expression Profiles from Laser Capture Microdissection, Transcript Amplification, and High-Density Oligonucleotide Microarray Analysis
Dr. Mark A. Watson, Assistant Professor of Pathology and Immunology, Washington University School of Medicine
Gene expression profiling using high-density oligonucleotide arrays is a powerful method to generate an unbiased survey of a cell's transcriptional landscape. Increasingly complex biological questions require that this approach be applicable to the small numbers of cells that are obtained from sources such as Laser Capture Microdissection (LCM) of solid tissues. We have demonstrated that two rounds of transcript amplification using a commercially available reagent set can generate accurate and reproducible gene expression profiles using high-density oligonucleotide microarrays, starting with as little as 10 nanograms of total RNA. Greater than 95% of all genes detected demonstrate less than a twofold difference in expression when independent tissue dissections of identical cell populations are compared. The accuracy and technical reproducibility of the method suggest that expression profiling using transcript amplification and high-density oligonucleotide microarrays can be used on a routine basis. We have begun to utilize this approach to characterize patterns of gene expression associated with the early progression of human breast cancer and to identify specific genes associated with this process.

4:30 Balanced PCR: A New Method for Retaining the Difference between Two PCR-Amplified Complex Genomes
Dr. G. Mike Makrigiorgos, Department of Radiation Oncology, Dana-Farber Cancer Institute and Harvard Medical School
Increasing emergence of genomewide analysis technologies frequently leads to a need to PCR-amplify entire genomes or cDNAs prior to performing comparisons among them. Amplification occurring in a nonlinear manner is a major problem with PCR. We have developed a new method, which overcomes the difficulty of nonlinear PCR-amplification of complex genomes and faithfully retains the difference among corresponding genes or gene fragments. It utilizes a simple principle-that by mixing the genomes PCR "loses" the ability to discriminate in any fashion among alleles and is applied by ligating specially designed composite linkers to the DNA populations to be compared. The ligated genomes are mixed and amplified in a single PCR reaction with a single primer common to both linkers, until the desired amount of final product is obtained. The original difference between any two genomic DNA fragments is faithfully retained for all genes simultaneously. High-throughput validation is performed using human prostate and lung cDNA applied on the Affymetrix Genechip® microarrays. Genes important to prostate cancer development are overestimated by more than a factor of ten when amplified via traditional PCR but correctly quantitated when amplified via balanced PCR. By removing nonlinearity from PCR amplification, the current balanced PCR approach releases the full potential of PCR and enables a range of genomic analysis technologies to be applied to minute amounts of starting DNA material.

5:00 A New, Novel Isothermal Linear Amplification (SPIA™) for mRNA Amplification and Gene Expression Profiling
Dr. Nurith Kurn, Chief Scientific Officer, NuGEN Technologies Inc.
We have developed a new, novel, and very rapid, isothermal, linear nucleic acid amplification method called SPIA™. This new method has been successfully applied to global expression profiling and quantitative gene expression analysis. The linear SPIA™ amplification of mRNA from very small total RNA samples (<20 ng) results in the generation of greater than 1,000-fold single-stranded DNA copies of each gene transcript while accurately maintaining the fidelity of representation of expression. This new method further incorporates novel approaches for labeling the amplified products, which are then suitable for global gene expression profiling analysis on microarrays, as well as for analysis of defined gene products by real-time monitoring methods (Real-Time PCR or TaqMan). The SPIA™ expression analysis method is very fast (about three hours), highly reproducible, robust, and simple to run.

8:00 Chair's Comments
Dr. Mark G. Erlander, Vice President and Division Manager for Arcturus Applied Genomics, Arcturus

8:05 Identification of Novel Molecular Signatures Discriminating Stage and Grade of Breast Cancer Using Laser Capture Microdissection
Dr. Mark G. Erlander
The integration of Laser Capture Microdissection (LCM) with microarray technology continues to be a powerful approach for identifying cell-specific expression within diagnostic-marker/signature discovery programs. However, the use of LCM in the clinical lab is in its infancy. Here we report, via LCM and microarrays, the identification of novel molecular signatures that discriminate stage and grade of breast cancer. Furthermore, we demonstrate the use of these signatures for predicting stage/grade of breast cancer cells via automated LCM of breast cells from cytological preparations.

8:35 Understanding Molecular Mechanisms Underlying Human Reproduction through Global Gene Expression Profiling
Dr. Renee A. Reijo Pera, Assistant Professor, Department of Obstetrics, Gynecology and Reproductive Sciences; Physiology and Urology Programs in Human Genetics and Cancer Genetics; University of California, San Francisco
Little is known of the genes required for the production of functional germ cells, the oocytes and sperm, in women and men. Even less is known of the genetic pathways required for activation of embryonic development, once egg and sperm fuse. We are examining the global gene expression patterns in human germ cells and early embryos to begin to identify and characterize critical genes for early human development.

9:05 PBK (PDZ-Binding Kinase) as a Tumor Marker
Ms. Susan M. Goldsworthy, Genomic Histology, GlaxoSmithKline
PBK is a protein that is overexpressed in tumor cells. Details on identification of the gene, linking it to carcinogenesis, analysis in preneoplastic lesions by LCM and QRT-PCR, and localization by immunohistochemistry, will be presented.

10:15 Gene Profiling of Precancerous Lesions in Mouse Colon Using Laser Capture Microscopy Combined with Linear Amplification of Total RNA
Dr. Daniel W. Rosenberg, Associate Professor of Medicine, and Investigator, Center for Molecular Medicine, University of Connecticut Health Center
Differential susceptibility to the colonotropic carcinogen, azoxymethane (AOM), has been described in A/J (sensitive) and AKR/J (resistant) mice. Although preneoplastic lesions, referred to as aberrant crypt foci (ACF), are formed within both susceptible and resistant strains, ACF that develop in A/J mice are more likely to progress to tumors. Interestingly, ACF from A/J mice are most dysplastic and are therefore thought to favor the formation of tumors. To enable further molecular characterization of ACF and to strengthen existing pathologic criteria, A/J and AKR/J mice were injected intraperitoneally with AOM (10 mg/kg body weight) once a week for six weeks. Frozen sections of distal colon were prepared at one, six, and nine weeks following treatment. Laser capture microscopy was used to procure individual populations of altered colon cells (as few as 50 cells per lesion). Using a microarray containing 15,000 mouse cDNA clones, mRNA expression patterns were analyzed after linear amplification of RNA extracted from laser-captured hyperplastic and dysplastic ACF from both strains, and microadenomas from A/J colons. To establish patterns of response within subsets of premalignant lesions and to help visualize the data in a logical way with a focus on biological response, we employed a novel adaptive centroid algorithm (ACA) for finding gene clusters with differential expression patterns between the various morphologically classified ACF. Our data indicate that a cDNA microarray approach, combined with the enormous sensitivity afforded by LCM-linear amplification methodology, provides a comprehensive overview of strain-specific and pathology-associated genetic alterations that occur during different stages of ACF development in the colon. This molecular profiling approach will greatly enhance our ability to predict the tumorigenic potential of preneoplastic lesions of varying morphology.

10:45 Gene Expression Analyses in the Central Nervous System: From Whole Brain to Single Cell
Dr. Bernd Meurers, Principal Scientist, Genomic Neuroscience, Johnson & Johnson Pharmaceutical Research and Development
Large-scale molecular profiling has become the method of choice to study complex biological systems. In the brain, where cells belonging to different functional systems are often closely associated, Laser Capture Microdissection (LCM) allowing the excision of single neurons greatly enhances the sensitivity and specificity of DNA array experiments. The different components of the technology as well as examples of potential biological applications will be presented.

11:15 Neurons That Matter: Profiling Specific Cell Types for Target Discovery in the Nervous System
Dr. Michael C. Ellis, Director, Genomics, Renovis, Inc.
The nervous system presents an enormous challenge for drug target discovery, as it contains more than twice the level of cellular complexity than the rest of the body combined. Renovis overcomes this barrier through technologies that provide access to the "neurons that matter"-the specific neuronal cell types that control particular behaviors and underlie various neurological or psychiatric diseases and disorders. Our research engine enables the labeling, manipulation, and isolation of these cell populations for both expression profiling and systematic biological validation. We will discuss data highlights from our neuropathic pain program that illustrate the dramatic increase in sensitivity observed when profiling isolated neuronal populations relative to heterogeneous tissue.

1:30 Chair's Comments
Dr. Jean Rossier, Professor of Biology, Ecole Supérieure de Physique et de Chimie Industrielles, and Director of the Neurobiology and Cellular Diversity Laboratory, CNRS

1:35 Transcriptome, Proteome, and Metabolome of a Single Cell: Harvesting with Patch-Clamp Pipettes
Dr. Jean Rossier
Although all cells of multicellular organisms have an identical genome, individual cells express a different set of genes resulting in a multiplicity of specialized cells expressing different functions. One of the present goals of biological research is to be able to study single cells and decipher in a single cell the set of genes transcribed in messenger RNA (transcriptome), the set of proteins translated (proteome), and the set of metabolites synthesized (metabolome). This task is difficult due to rarity of materials in a single cell. With multiplex RT-PCR, expression of 100 different genes can be monitored simultaneously. The transcriptome, proteome, and metabolome analyses at the single-cell level represent a new frontier in biology and are essential to deciphering the heterogeneity and the function of the neuronal network of the brain. Diversity of neocortical interneurons at the gene-expression level is associated with different functional properties. Some interneurons behave like coincidence detectors and are thus important to control synchrony of brain electrical waves. Other interneurons control brain metabolism and blood perfusion making these neurons good candidates as targets for the development of new drugs in neuropsychopharmacology.

2:05 Nanoarrays for Proteomics
Dr. Eric Henderson, Chief Science Officer, BioForce Nanosciences, Inc.
Nanoarrays occupy 1/10,000 of the area of typical microarrays. This allows researchers to visualize over a thousand protein domains in a single optical microscope image and to realize the benefits of using novel read-out methods such as atomic force microscopy (AFM). With nanoarrays, it is feasible to carry out rapid, multiplexed molecular analyses from samples as small as a single cell, without signal amplification

3:00 Targeted Proteomic Analyses of Small Cell Groups
Dr. Howard B. Gutstein, Associate Professor, Departments of Anesthesiology and Molecular Genetics, University of Texas-MD Anderson Cancer Center
A major issue in proteomic analysis is the ability to profile protein expression in specific cell groups or cells expressing specific markers. We have developed techniques to permit accurate protein profiling of cells from specific brain regions using Laser Capture Microdissection (LCM) coupled to two-dimensional gel electrophoresis (2DE). Unlike the situation for mRNA expression, conventional tissue staining (and several unconventional stains) greatly reduces protein recovery from samples. We have refined the technique of "navigated" LCM, in which an adjacent stained section is used to guide the capture of specific cells from an unstained section, so there is minimal loss of protein expression using this technique. We have also modified conventional immunostaining protocols such that proteins from cells expressing specific markers can be recovered with approximately 90% fidelity. These techniques are currently being applied in our laboratory to address questions of interest in the field of drug addiction. These methods should also have broad applicability to questions of biological and pharmacological interest.

3:30 Padlock- and Proximity-Probe Ligation Assays for Localized Detection of Single Molecules and for Parallel Analysis of Genes, Transcripts, and Proteins
Dr. Mats Nilsson, Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University
We have developed two complementary technologies for highly specific and sensitive localized detection of nucleic acid and protein molecules in cells and tissues. The first technology, based on padlock probes, can be used for in situ genotyping of SNPs in DNA or direct analysis of SNPs of RNA sequences. Padlock probes can be combined in large numbers for parallel analysis of genes and transcripts. We are currently adding tens of probes to individual genomic DNA samples, and we have experimental evidence that thousands of probes can be analyzed in parallel. Proximity-probe ligation, which makes use of pairs of oligonucleotide-tagged protein affinity reagents such as antibodies or DNA aptamers, can be amplified by real-time PCR, thereby allowing quantitative and sensitive protein analysis over a broad range of concentrations. Both padlock- and proximity-probe ligation reactions can be detected in situ using rolling circle replication to study the localization of individual DNA, RNA, or protein molecules. We have further developed an enhanced rolling-circle replication reaction that is as sensitive as PCR but works better for amplification of large numbers of circularized padlock or proximity probes. The quantitative resolution is high with a CV of 2% when the reaction is followed in real time using a modified molecular beacon design.

PREMIER SPONSOR BIOGRAPHY
	Arcturus provides microgenomics products for cell-specific molecular analysis - integrated instruments and reagents featuring the PixCell IIe Laser Capture Microdissection (LCM) System - and maintains a state-of-the-art applied genomics laboratory.