PRE-CONFERENCE SHORT COURSE*
MONDAY, MARCH 23
8:00-8:30 Registration for Pre-Conference Short Course
8:30-1:00 Pre-Conference Short Course*
(*Separate Registration Required)
microRNA IDENTIFICATION, PROFILING AND
VALIDATION TECHNIQUES
8:30-8:35 Chairperson’s Opening Remarks
Don A. Baldwin, Ph.D., Director, Penn Genomics Facility, University of Pennsylvania
8:35-9:00 Multiplex Assays for microRNA
Don A. Baldwin, Ph.D., Director, Penn Genomics Facility, University of Pennsylvania
The rapid development of several genomics platforms for microRNA discovery and detection has created opportunities for translation to biomarker applications. Current models for microRNA production and function will be reviewed, as well as enabling technologies for identification, extraction, amplification and labeling of microRNAs. Detection assays range from high-throughput single target methods to custom multiplex panels to comprehensive microarrays, genomic tiling arrays and next generation sequencing. microRNA examples will be used to illustrate the potential of these assays for biomarker development, including cancer profiling from serum and archival tissue samples.
9:00-9:25 Technology Development for Low-Cost and High-Throughput Sequencing of microRNAs
Francois Vigneault, Ph.D., Church Laboratory, Department of Genetics, Harvard Medical School
microRNAs (miRNAs) are endogenous 21–23-nt noncoding RNAs that post-transcriptionally repress gene expression by binding to 3’-untranslated regions of target mRNAs in a sequence-specific fashion to impair mRNA translation and/or stability. They are critical regulators of most major cellular pathways such as cellular differentiation, apoptosis, and metabolism. Although most studies rely on the detection of previously reported miRNAs, the most powerful approach to identify and quantify expression of new miRNAs remains direct cloning and sequencing. To this extent, we have developed a procedure to facilitate miRNA capture via ligation by barcoding samples, thus allowing multiplexing and considerably minimizing the per-sample cost on next-generation sequencing platforms.
Sponsored by
9:25-9:55 Microfluidic Technology Enabling Advances in microRNA Research
Christoph Eicken, Ph.D., Head, Technical Services Microarrays, LC Sciences LLC
An advanced biochip system based on a flexible microfluidic technology designed to stay current with the rapidly evolving microRNA field, and perform diverse small RNA discovery experiments is presented. This technology’s unique flexibility allows for miRBase synchronicity and design of customized biochips adapted to each researcher’s specific needs. Applications featuring disease marker discovery, drug treatment, microRNA target screening, and small RNA discovery are highlighted.
9:55-10:10 Coffee Break
10:10-10:35 A microRNA qRT-PCR Assay that Differentiates Pancreatic Ductal Adenocarcinoma from Chronic Pancreatitis
Anna Szafranska-Schwarzbach, Ph.D., CLIA Laboratory Supervisor, Pharmacogenomics Services, Asuragen, Inc.
Using microarray and qRT-PCR platforms we identified miR-196a and miR-217 as the top biomarker candidates that distinguish pancreatic ductal ade-nocarcinoma (PDAC) from chronic pancreatitis. The qRT-PCR assay developed using this microRNA (miRNA) signature was validated using formalin-fixed, paraffin embedded (FFPE) pancreatic blocks and achieved 95.24% sensitivity and 94.87% specificity. Early feasibility experiments showed that the assay can also be successfully used to identify PDAC in low tissue yielding clinical specimens, such as fine needle aspirate biopsies. In addition, interrogation of microdissected populations of normal, pre-malignant and malignant cells revealed that miR-196a is specific to PDAC cells and can be detected as early as in PanIn-2 precursor lesions. Our ongoing efforts will assess whether elevated expression of miR-196a in pancreatic tissue may enable earlier identification of patients at high risk to develop PDAC in the future.
10:35-11:05 Sponsored Presentation (Opportunity available)
Contact Jon Stroup, Manager, Business Development, at jstroup@healthtech.com or 781-972-5483.
11:05-11:30 Stem-Loop RT-qPCR miRNA Profiling: About Single Cells, Normalization and Biomarkers
Pieter Mestdagh, Ph.D., Center for Medical Genetics Ghent, Ghent University Hospital
MicroRNAs are an emerging class of small non-coding RNAs implicated in a wide variety of biological and cellular processes. Research in this field is accelerating and the growing number of miRNAs emphasizes the need for high-throughput and sensitive detection methods. The Megaplex stem-loop RT-PCR procedure in combination with limited-cycle pre-amplification is a powerful method for miRNA expression profiling in both large and small cell populations capable of covering the majority of human miRNAs. Its superior sensitivity allows for accurate miRNA profiling of samples yielding only minimal amounts of RNA such as small tumour biopsies or patient serum. Given a proper normalization strategy, reliable miRNA profiles can be obtained from these samples enabling the identification of prognostic and diagnostic miRNA signatures or novel biomarkers.
11:30-11:45 Break
11:45-12:10 Analysis of Transrenal microRNA for Measurement of in vivo Cell Death and Potential Diagnostic Applications
Samuil Umansky, M.D., Ph.D., Chief Scientific Officer, Xenomics, Inc.
Cell death is involved in pathogenesis or accompanies numerous diseases and its detection in vivo would be useful for screening, diagnostic and monitoring purposes. Xenomics has developed a new technique for isolation of cell-free nucleic acids and demonstrated the presence in urine of microRNA specific for organs located outside of urinary system, e.g. brain-, heart-, liver-, tumor- or placenta-specific microRNA. Concentrations of neuron-specific miRNA increases in urine of patients with acute and chronic neurological disorders and can be used as a biomarker of in vivo neuronal death.
12:10-12:35 Identifying microRNA Regulators of the Cell Cycle
Michele Cleary, Ph.D., Director, Biology, Rosetta Inpharmatics LLC, a wholly owned subsidiary of Merck & Co., Inc.
One of the major challenges in understanding microRNA function is the identification of the target or targets responsible for an observed phenotype. Although target prediction algorithms are fairly good, there is disagreement among the various approaches and many targets predicted on the basis of 3’ UTR seed region complementary are not confirmed in microRNA functional studies. We use microarray-based gene expression profiling as a first step in determining candidate targets for microRNAs. This approach yields rich sets of genes that are influenced by perturbations in microRNA levels and which include primary and secondary targets. Annotation of these gene sets can often provide an understanding of microRNA function. We also combine microRNA and mRNA profiling of disease and normal tissue to establish correlations that can provide further insight to microRNA. By use of these integrated approaches, along with molecular and phenotypic analysis, we have identified and characterized several microRNAs that regulate cell cycle progression, some of which play a role in the p53 DNA damage response.
12:35-1:00 Micromanagers of Metastasis
Li Ma, Ph.D., Life Sciences Research Foundation Fellow, Weinberg Lab, Whitehead Institute, Massachusetts Institute of Technology
MicroRNAs (miRNAs) are small non-coding RNA molecules that suppress gene expression by interacting with multiple target mRNAs. In an initial qPCR-based screen for miRNAs differentially expressed in human breast cancer cells, we identified several most significantly deregulated miRNAs. The subsequent functional studies of miR-10b validated its candidacy as a mechanistically important miRNA, as demonstrated by functional experiments showing that overexpression of miR-10b in otherwise-non-metastatic breast tumors initiated tumor invasion and distant metastasis in xenograft models. These findings provide the first evidence that over-expression of a miRNA can contribute to the development of metastasis. Moreover, our recent studies of a second candidate miRNA that stood out in the initial screening revealed that this miRNA can regulate metastasis through a unique signaling pathway.
1:00 Close of Short Course