yale 2009 presentation part2
TRANSCRIPT
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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Challenges and Solutions
•! 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
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1
3
2
0
20
40
60
80
100
120
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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
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Knockdown in E-selectin assay HUVEC cells-48hr transfection
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Challenges and Solutions
•! 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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Challenges and Solutions
•! 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
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26
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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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•! 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
Sample
Fold
Reduction
2
2.5
1.5
1
Active Threshold
(FD>=2)
Median
Advanced Hit Selection
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Fold
Reduction
2
2.5
1.5
1
Active Threshold
(FD>=2)
Median
•! 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
Advanced Hit Selection
Sample
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Fold
Reduction
2
2.5
1.5
1
Active Threshold
(FD>=2)
Median
•! 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
Advanced Hit Selection
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
Challenges and Solutions
•! 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
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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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