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NHLBI Genomics Core Facility. 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. Primary Goals.

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Nhlbi genomics core facility

NHLBI Genomics Core Facility


Nhlbi genomics core facility

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


Primary goals
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


Nhlbi genomics core facility

Resources in the Core

Nanodrop spectrophotometer

Agilent’s Lab on a chip



Nhlbi genomics core facility

Hyb Oven Fluidic Station Scanner

Peg Arrays

Human U133A gene chips only

Scanner


Nhlbi genomics core facility

ABI 7900 – Sequence Detection System

Q-PCR

Perkin Elmer ScanArray 5000XL

2 color spotted arrays


Application software in the core
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


Nhlbi genomics core facility

Expression Profiling Workflow

(Spike in

labeling controls)

2 µg

(Spike in

hyb controls)


Nhlbi genomics core facility

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


Nhlbi genomics core facility

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


Nhlbi genomics core facility

Basic Level of Service

  • Online access to project data


Nhlbi genomics core facility

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


Nhlbi genomics core facility

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


Nhlbi genomics core facility

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


Core policies
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


Contact information
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