Yale 2009 presentation part2

Projects

•! Grant Applications

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Grants & Contracts •! NIH Grants Submitted:

o! Assay Development for High Throughput Molecular Screening (R21)

o! Recovery Act Limited Competition: High-End Instrumentation Grant Program (S10)

•! Collaboration Grants: o! Fidelity Non-Profit Management Foundation – 5%FTE (Dr. Stern

and Dr. Bosenber) - Awarded

o! NIH RC1 Grant – 10% FTE (Dr. Cantley)

•! Grants submitted to include funding for our Services: o! Dr. Richard Sutton

o! Dr. Scott Small

o! Dr. Jian Cao

o! Dr. Harold Smith

o! Dr. Jack Rogers

o! Dr. Dan Wu

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Challenges and Solutions

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•! Transfections

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siRNA Transfections: Variations Tested

•! Cell Types –! HEK293 –! Hela –! HUVEC

•! Transfection Reagents –! RNAiMAX –! Dharmacon #1 –! Icafectin –! Lipofectamine 2000 –! N-TER peptide

•! Protocols –! Reverse Transfection –! Forward Transfection –! Dry-Down Transfections –! Antibiotics Usage –! Media Optimization

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Variation among Fields: Solid-State Reverse Transfection

Field Placement in Well

4

1

3

2

0

20

40

60

80

100

120

140

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

GAPDH siRNA Mock Transfected

GA

PD

H C

yto

pla

sm

ic In

ten

sit

y -

% o

f C

on

tro

l

Row

GAPDH Knockdown in Hela Cells

Field 1

Field 2

Field 3

Field 4

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Solid State Knockdown in HUVEC

No TNFa Control siRNA Mock Transfected

E-sel knockdown 2 E-sel knockdown 1

•!Transfection complexes were added to plate and dried down

•!Cells were plated after drying

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Reverse Knockdown in HUVEC Normalized data for RNAiMax transfections

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Measuring Knockdown of mRNA Using RT-PCR 24hr transfections in HUVEC

2.2

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Control siRNA TNFR1 E-selectin

E-selectin Assay 48 hr knockdown

0

100

200

300

400

500

600

700

Neg Control Pos Control E-selectin TNFR1

Cy

top

lasm

ic In

ten

sit

y

Knockdown in E-selectin assay HUVEC cells-48hr transfection

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•! Automation and Imaging Hardware

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Infrastructure Break-In Issues

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Multidrop 384 Break-In

Row A

Row G

Row M 0

5000

10000

15000

20000

25000

30000

1 3 5 7 9 11 13 15 17 19 21 23

Flu

ore

scen

ce

Column No.

FITC Test 384-Well Plate

25000-30000

20000-25000

15000-20000

10000-15000

5000-10000

0-5000 Row A

Row G

Row M 0

5000

10000

15000

20000

25000

30000

1 3 5 7 9 11 13 15 17 19 21

Flu

ore

scen

ce

Column No.

FITC Test 384-Well Plate

Average: 18,300

S. D.: 2,600 (14%) Average: 21,400

S. D.: 880 (4%)

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•! Informatics and Advanced Data Analysis

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Data QC through XE Runner

-!2 plates from same assay run

-!Invalidated 2 wells on the first plate due to contamination issues

-!Invalidated the second plate in its entirety due to poor staining on the plate

-!Can also invalidate wells based on quantitative thresholds, shown below (e.g. invalidate

wells with less than 10 cells analyzed)

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Wells containing siRNA under investigations

3 x Positive Control Standard Deviation + 3 x Negative Control Standard Deviation

1 - | Positive Control Mean – Negative Control Mean|

QC through Plate & Run Stats

Positive

controls Negative

controls

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Biological Process Annotation

GO Biological Process # Genes

signal transduction 28

regulation of transcription, DNA-dependent 26

ion transport 18

protein amino acid phosphorylation 18

transcription 18

ubiquitin-dependent protein catabolic process 18

multicellular organismal development 15

cell adhesion 14

transport 11

G-protein coupled receptor protein signaling pathway 10

intracellular protein transport 10

protein transport 10

proton transport 10

28

26

18

18

18 18

15

14

11

10

10

10 10

signal transduction

regulation of transcription, DNA-

dependent

ion transport

protein amino acid phosphorylation

transcription

ubiquitin-dependent protein catabolic

process

multicellular organismal development

cell adhesion

transport

G-protein coupled receptor protein

signaling pathway

intracellular protein transport

protein transport

proton transport

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Advanced Hit Selection

•! Interrogate hit genes that: –! Group in the most

represented clusters AND/OR

–! Group in the most relevant functional cluster, i.e., containing gene(s) of interest

–! Include marginal hits that fit other criteria

Fold

Reduction

2

2.5

1.5

1

Active Threshold

(FD>=2)

Median

Sample

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Sample

Fold

Reduction

2

2.5

1.5

1

Active Threshold

(FD>=2)

Median


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Fold

Reduction

2

2.5

1.5

1

Active Threshold

(FD>=2)

Median






Sample

49

Fold

Reduction

2

2.5

1.5

1

Active Threshold

(FD>=2)

Median






Sample

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Advanced Data Analysis

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•!Clustering •!Pathways •!Networks •!Compounds •!Diseases •!Tissues •!Gene Profiler •!Gene Set Overlap •!Splice Variants •!Inferred Hits •!Follow-up Screens


•! Client Acquisition, Partnerships, and Management

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Contact List generated

•!Either from Marketing Company contacts or HTCB data mining

Email blast with follow-up phone calls

•!Interested potential clients receive personal communiqué from HTCB suggesting meeting

Interested potential clients and HTCB meet (phone or in-person) and discuss client needs and HTCB strengths

•!If external funding is needed, HTCB provides boilerplate material to support potential client

Agreement drafted between HTCB and client

•!Work begins

Client Acquisition Workflow Overview

0

5

10

15

20

25

% Feb 2009 (2458)

Mar 2009 (2747)

June 2009 (1159) Responses to Marketing Emails

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Website Benchmarking: January – June, 2009 Criteria Yale HTCB Website Benchmark Comparison*

Visits 2,740 +324%

Pageviews 11,849 +751%

Pages / Visit 4.32 +101%

Bounce Rate 37.15 -25%

Average Time On-Site

3:04 +249%

Percent New Visitors 54.34 -18%

*Benchmarked to Biological Sciences sites of similar size by Google Analytics

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Yale-Outreach •! Yale Daily News •! Yale Bulletin •! Yale School of Medicine

Magazine •! MB&B Presentation •! Cell Biology Progress

Reports •! RNAi Summits •! HCA Conference •! Free screens

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Antibody and assay

development

Imaging technology

Image management

Database integration

RT-PCR

HTCB Industrial Partnerships

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Project Management •! ISO 9000 – Quality Management Principles

•! “Say what you’re going to do” •! “Do what you say” •! “Prove it”

–! Client-Focus –! Involved Leadership –! Staff Involvement in all aspects –! Process Approach –! Systems Approach to Management –! Continual Improvement –! Factual Approach to Decision

Making –! Mutually Beneficial Supplier

Relationships

Client / Collaborator

Assay Principle

Introduce

Assay Development

Assay Parameters

Define

Cell CultureHTSInformatics Cells

Culture

Imaging Plates

Produce

Images and Data

Acquire

Hit Report

Generate

Deliver Report

7.2.2 Review of Requirements Related to the Product

7.3.5 Design and Development Verification

8.3 Control of Nonconforming Product

7.3.4 Design and Development Review

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Summary and Future Directions

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Summary

•! Yale HTCB has, in its first year of operation, overcome infrastructure, location, and novel application challenges to implement a screening facility staffed by experts operating cutting-edge equipment for the purpose of providing screening services to the Yale Community and beyond.

•! The shift to an RNAi-exclusive focus has led Yale HTCB to concentrate on building core competencies, reaching out to interested parties, and acquiring suitable projects.

•! Center infrastructure, wholly in-place and functional on April 1, 2009, is nearing the end of its break-in phase. Center staff is eager to work on the diverse internal and external projects. The project pipeline is full and marketing efforts to keep it so are progressing.

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Future Plans and Potential Hurdles

•! Yale HTCB shall continue to refine its biological, high throughput automation, and informatics expertise and toolsets.

•! Yale HTCB shall complete all projects currently in the pipeline and shall continue its marketing and outreach efforts to keep its pipeline full.

•! Yale HTCB will host a High-Content High Throughput Cell-Based Screening Symposium.

•! Yale HTCB must plan for a disruptive move out of its present space into its permanent home.

•! Yale HTCB must continue to overcome the challenges presented by its remote location to New Haven and the Yale scientific community.

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Yale 2009 presentation part2

Science

Transcript of Yale 2009 presentation part2