NHLBI Genomics Core Facility

1. NHLBI Genomics Core Facility

2. New Organizational Structure Bio-Informatics Wet lab Peter Munson, Ph.D Director, MSCL, CIT Nalini Raghavachari, Ph.D Xiuli Xu, Ph.D Delong Liu, Ph.D Tony Cooper Jim Taylor, M.D MSCL Staff, CIT Kim Woodhouse Hangxia Qiu, Ph.D

3. Primary Goals • To provide investigators with high quality, state-of-the-art gene expression profiling & genotyping services in a timely fashion using the affymetrix platform • rigorous standardization of protocols and multiple quality control checks • To provide streamlined data analysis and identify signature genes • Application of complex statistical tools

4. Resources in the Core Nanodrop spectrophotometer Agilent’s Lab on a chip

5. GCAS Robot for sample processing

6. Hyb Oven Fluidic Station Scanner Peg Arrays Human U133A gene chips only Scanner

7. ABI 7900 – Sequence Detection System Q-PCR Perkin Elmer ScanArray 5000XL 2 color spotted arrays

8. Application software in the core • GCOS - Data collection • MSCL Toolbox - Normalization/Analysis • Jmp - Data Set Analysis • Genespring - Data Set Analysis • Partek Pro - Normalization/Analysis • Ingenuity - Pathway analysis • Metacore - Pathway Analysis

9. Expression Profiling Workflow (Spike in labeling controls) 2 µg (Spike in hyb controls)

10. Genotyping Workflow 250 ng Genomic DNA Restriction Digestion Day 1 Nsp1 Nsp1 Nsp1 Nsp1 200 – 1100 bases Single Primer Amplification Day 2 Day 3 Hyb, Wash, and Stain. Scan and Data Analysis Fragmentation and Labeling

11. Data Flow and Analysis Inage and QC data are checked Retrieval of data from NIHLIMS Normalize & Transform data Principal Component Analysis Statistical Tests - t-tests, ANOVA, Cluster analysis 1 2 3 Multiple comparison corrections Apply FDR and FC filters select gene lists Gene Ontology, Pathway analysis, Bio-Informatics Scanner GCOS Client Signet Investigator NIHLIMS Backup servers DVD archives of data

12. Basic Level of Service • Online access to project data

13. Standard Level Consultation on project Recommendations on RNA isolation Amplification of RNA ~ 3000cells/3-5 ng of RNA Simple statistical analysis, gene selection Pathway Analysis (GSEA, Genego, GoScan, GoMap) Help with Publication Target(s) identification

14. Advanced Analysis Help with Publications Taqman Analysis (qPCR) Target(s) identification Collaborate on experimental design co-authorship in publications RNA isolation Amplification of RNA Novel / Complex Statistical Methods (Munson’s group) Novel Pathway Analysis

15. New Service Initiatives • Taqman Low Density Arrays (can analyze 8 samples for 24 different genes) • Exon and Tiling Array Mapping • Method development for the isolation and analysis of picogram amounts of RNA in collaboration with bio-tech companies • Service for non NHLBI investigators at a nominal cost

16. Core Policies • Investigator has to provide chips and samples to the core (We need at least 2µg total RNA in 4µl volume, 250ng genomic DNA) • Submitted RNA/DNA samples should be of good quality • Description of project / sample information • It is advisable to have an n of 5-6 for each group to have a good statistical power for analysis • If you have only one or 2 samples per group, the core wont be responsible for data quality • Turn around time is 2 weeks since the receipt of samples • You will be notified by email the status of your project and upon completion will send you QC data • If a problem arises with the analysis of one or more of your specimens, you will be contacted immediately

17. Contact Information • Mark Gladwin – 301-435-2310 mgladwin@nhlbi.nih.gov • Nalini Raghavachari – 301-435-2304 nraghavachari@cc.nih.gov • Tony Cooper – 301-496-8123 cooperro@mail.nih.gov • Xiuli Xu 301-402-2102 xux3@nhlbi.nih.gov • Hangxia Qiu 301-451-6253 qiuh2@mail.nih.gov • Website NHLBI/Vascular Medicine Branch/Genomics Core contacts, protocols, status of projects, meetings, tutorials