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. The Core became an integral part of Pulmonary and Vascular Medicine Branch in 2006 and since then it has been diligently organized into 2 major scientific teams comprising of multidisciplinary scientists who function as biologists in the Wet lab or Statisticians performing data analysis and bioinformatics. These 2 teams along with other Statisticians, System Administrators in CIT and investigators meet on the first and 3rd Fridays of every month from 3-6PM to discuss new projects, new technologies, Data Storage, Analysis and other issues related to core work. Periodically, we bring in representative from speakers from commercial organizations to address new technologies and data analysis programs/software. 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 Gold 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 (A). Method Development in collaboration with NUGEN Inc - To Isolate RNA from cells as few as 1-10 cells, RNA amplification and gene expression profiling/ alternative splicing events using exon arrays (B). Study of Endothelial cell Dysfunction in Vascular Diseases - Collection of endothelial cell by brushing / scraping cells off aorta enrichments of ECs using magnetic beads, RNA amplification and gene chip studies (C). Alternative splicing events using exon arrays (D). Technology development for MicroRNA profiling -The core has optimized methods to isolate total RNA enriched with MicroRNA from whole blood, isolated blood cells, and labeling of microRNA with biotin for gene chip analysis 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 10 publications in Cell, Nat. Genetics, Genome Res, Physiol genomics, PNAS Std and collaborative Service 8 publications in Circ, Circ Res, ATVB, J Exp Med, Blood, JBC, BMC Genomics, Free Rad Biol Res 1 book chapter, 1 Journal Commentary 2 manuscripts in review and 4 in preparation Collaborative studies completed with CCMD (Nalini Raghavachari served as Associate investigator in the following projects) 1) Atorvastin Therapy to improve endothelial function in sickle cell disease microarray analysis has been completed and the data is being analyzed now. 2) Endothelial dysfunction in HIV associated pulmonary hypertension A pilot study has been completed with samples collected in NIH and the data is being analyzed now. The core has also provided service to 6 major projects from non NHLBI institutes such as NEI, NICHD and NINDS in the last 2 years.The core now has several Ongoing Major Collaborative Clinical Projects (1). SNP analysis, Homozygosity Scan and Copy Number Variation analysis on Ebstein family samples (>100) from the island of Pemba - A Project of Dr. Cecilia Lo (2). 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 (3). 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 Collaborative Study with INOVA Hospital in Fairfax and Dr. Roberto Machado (4). The Effect of CCR5 Inhibition on Pulmonary Sarcoidosis to examine the transcriptomes in both the BALF and thoracic lymph node compartments, using RNA amplification techniques coupled with high density gene expression arrays. Our primary analysis will focus on the lymph node tissue and flow sorted CD4+ T cells;the latter will limit the confounding effects of cell differential on transcriptional analysis. Gene enrichment set analysis of CCR5 canonical pathways will increase our power for primary assessment of the role of CCR5 activation in both compartments. Project of Dr. Joseph Fontana (5). Clock Gene Regulation Studies on whole blood to evaluate the influence of hyper and hypocortisolism. Project of Dr. Lynette Nieman in NICHD The core director has received the following Bench to Bedside Awards jointly with other investigators. (A). Gene Expression Profiling to Predict Sickle Cell Anemia Sub-Phenotypes 2009 (B). Endothelial dysfunction in primary pulmonary hypertension - 2007
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