The NHLBI Genomics Core facility provides a full spectrum of services using affymetrix platform for parallel analyses of genomes and their expression. Applications of this technology include global gene expression profiling, exon analysis to study alternative splicing events, microRNA analysis, and large scale SNP genotyping using the SNP 6.0 chips and mapping sites of protein/DNA interaction using tiling arrays. The primary goals of the core are (1). To provide investigators with high quality, genome analysis service in a timely fashion by rigorous standardization of protocols and multiple quality control checks and (2). To provide streamlined data analysis for identification of signature genes and biomarkers by the application of complex statistical tools. In addition, investigators with high throughput projects are supported by the robotics infrastructure within the facility. Overall governance of the core is performed by the Core steering committee Head Dr. Keji Zhao and the steering committee members Dr. Richard Cannon, Dr. Michael Sack, Dr. Warren Leonard and Dr. Adrian Wiestner provide suggestions to improve the operation of the core. Our core has a gene chip array station GCAS, a high-throughput and completely automated solution for large scale expression profiling projects using array plates. Components of GCAS system include Sciclone robotic station, array plate that contains 96 cartridge arrays and the new type of scanner with the capacity to process 96 arrays per run. SciClone robotic station performs automatic target preparation, hybridization and stain/wash steps of expression profiling process. Our core is one of the first few sites where the GCAS prototype was installed. We are capable of processing two 96 well plates/192 samples per day in a highly reproducible manner and have had very few problems in the operation of the robot. Samples processed by the GCAS can either be used on individual cartridge arrays or Peg plates for hybridization. Annually, our core provides microarray analysis for 60 different projects involving 1100-1200 gene chips as both standard and collaborative services. In some of these collaborative projects, the core helped the investigator design experiments, provided technical help in collecting samples, RNA isolation, RNA amplification with as little as 3-5 ng total RNA and Target labeling for gene chip analysis. 97% of these projects were gene expression analysis while 3% of the studies were on tiling arrays and SNP analysis. The statistical team provided extensive help in identifying differentially regulated genes, their biological functions and pathway analysis. Less than 5% of the projects are self analyzed by the investigator. With the amount of chip usage in the core, we have been participating as platin club member in the Affymetrix Core lab Program. With this club membership, we have been participating as a beta tester on new products, discounted pricing on instruments, software and seminars and web communications. The core is actively involved in the following research activities 1.Development of method for single cell transcriptome analysis. Evaluated a new technology in collaboration with Nugen for studying single or few cells without RNA isolation for transcriptome analysis. We collected single oocytes from wild type and knockout mice (Dr. Manganiello) and compared the differential expression of genes from single oocytes to 5 and 50 oocytes. This data was presented at the ABRF meeting in 2011 as part of the microarray research group presentation. Currently working on the transcriptome analysis of single hematopoietic cell and differentiated cells by QPCR and microarrays without RNA isolation. 2. Implications of RNA quality based on RIN for arrays analysis and applicability on FFPE samples This was a collaborative project with ABRF members . RNA samples with RIN at 2, 4 and 8 were analyzed by different labs on microRNA chips. This work was presented at the ABRF meeting in 2011. We are currently working on evaluating synthetic miRNA on differently platforms to evaluate their efficiency. 3. Transcriptome Profiling and Sequencing of differentiated Human Hematopoietic Stem cells Reveal Lineage Specific Expression and Alternative Splicing of Genes. This project was completed and the manuscript is in press in Physiological Genomics. 4. Performed a pilot study comparing exon arrays to RNAseq technology A Systematic Comparison of Exon Arrays to RNA-seq technology in Unraveling Sickle Cell Transcriptome. A manuscript is ready for submission. 5. Currently working with ABRF/FDA sequencing control consortium on the evaluation of technologies for RNAseq and library construction. Single cell transcriptome analysis by next gen sequencing will also be evaluated using one direct amplification system with protocol modifications by the core . 6. Working out details with Caliper/ Nugen for automation of library construction for RNAseq using the robot. This service is planned to be offered in the next fiscal year. Training Activities: The core trains students and fellows in the use of Qiacube for RNA/DNA isolation, Agailent bioanalyzer for measuring and checking the quality and ABI 7900 for QPCR assays. We also demonstrate how the arrays process is done starting from target labeling to getiing the raw data from the scanner. We also train investigators on data analysis and pathway analysis using Genespring, MSCL Toolbox, Metacore, and Ingenuity Pathway Analysis programs. (1). The SABRe CVD Initiative: Systems Approach to Biomarker Research in NHLBIs Framingham Heart Study.
Specific aims of the study are as follows: A. To characterize the molecular signatures of clinically important diseases in Atherosclerosis and Metabolic syndrome risk factors B. To use high-throughput technology to measure thousands of gene expression biomarkers with Exon arrays. Significant accomplishments 1. We have completed a feasibility study on 200 samples to identify the best sample type and the chip type for the large collection of samples. A manuscript has been written on the data generated from this study 2. Completed CVD case control study on 250 cases and 250 controls. Data is being analyzed now. 3. Completed CVD incident study on 223 cases and controls Data analysis is underway on this set of samples 4. Completed the chipping process on 2700 offspring samples 5. Currently chipping the GENIII samples . Completed Bench to Bedside Project in collaboration with INOVA hospital in fairfax Evaluation of the Platelet and PBMC Transcriptome in Pulmonary Arterial Hypertension to identify biomarkers in pulmonary arterial hypertension and to unravel the mechanism of endothelial dysfunction in PAH. A manuscript entitled A peripheral blood mononuclear cell molecular signature identifies patients with pulmonary arterial hypertension has been prepared for submission. With the service provided to the NIH investigators, the outcome with respect to Peer reviewed publications is as follows Basic service Acknowledgements to the core 15 publications in Cell, Nat. Genetics, Genome Res, Physiol genomics, PNAS Std and collaborative Service 2 book chapterS, 3 manuscripts in review and 2 in preparation Conference Presentations: 1. Whole transcriptome analysis of cd34+ hematopoietic stem cells during lineage specific cell differentiation: toward a molecular portrait of human hematopoiesis. Nalini Raghavachari Poster Presentation HGM 2011, Dubai. 2. Evaluation of One Direct amplification systems for single cell transcriptome analysis Nalini Raghavachari, MARG, ABRF February 2011. 3. Characterization of microRNA expression profile in platelets in sickle cell disease : American society of Pediatric Hematology-Oncology, Montreal, Canada;April 2010;
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