NHLBI Genomics Core Facility

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

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 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 To provide streamlined data analysis and identify signature genes  Application of complex statistical tools

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

GCAS Robot for sample processing

Peg Arrays Human U133A gene chips only Hyb Oven Fluidic StationScanner Scanner

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

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

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

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

Backup servers Scanner NIHLIMS GCOS Client Signet Investigator 123 DVD archives of data Inage and QC data are checked Retrieval of data from NIHLIMS Normalize & Transform data Principal Component Analysis Statistical Tests - t-tests, ANOVA, Cluster analysis Multiple comparison corrections Apply FDR and FC filters select gene lists Gene Ontology, Pathway analysis, Bio-Informatics Data Flow and Analysis Data Flow and Analysis

 Online access to project data Basic Level of Service Basic Level of Service

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) Standard Level Target(s) identification Help with Publication

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

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

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) 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 Submitted RNA/DNA samples should be of good quality Description of project / sample information 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 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 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 Turn around time is 2 weeks since the receipt of samples You will be notified by the status of your project and upon completion will send you QC data You will be notified by 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 If a problem arises with the analysis of one or more of your specimens, you will be contacted immediately

Contact Information Mark Gladwin – Mark Gladwin – Nalini Raghavachari – Nalini Raghavachari – Tony Cooper – Tony Cooper – Xiuli Xu Xiuli Xu Hangxia Qiu Hangxia Qiu Website Website NHLBI/Vascular Medicine Branch/Genomics Core contacts, protocols, status of projects, meetings, tutorials