1 A Decade of Microarrays ACGT Microarray Facility Nicky Olivier.
-
Upload
gage-trott -
Category
Documents
-
view
223 -
download
1
Transcript of 1 A Decade of Microarrays ACGT Microarray Facility Nicky Olivier.
1
A Decade of Microarrays
ACGT Microarray Facility
Nicky Olivier
In the beginning
● Institutions involved: ● UP & CSIR – Shared start-up costs
● Physical location: Agriculture Building● Special installation required
● Department Plant Science and FABI● Equipment - June 2001
● Setting up and validation● Core service: July 2002
2
People
• Director– Prof Dave Berger
• Scientific Officers– Danie Theron (2001-2006)– Nicky Olivier (2006-present)
• Statisticians– Loveness Dzikiti– Nanette Coetzer
3
Advisors
Prof Jane Morris (ACGT)
Prof Henk Huismans (UP)
• Equipment and hardware acquisitions
• Approval of projects
• Approval Annual Financial Statements
• Future plans for Facility
4
Start-up Instrumentation 2001
5
Molecular Dynamics GenIII Spotter
Genepix 4000B Scanner532 nm / 635 nm
Instrumentation 2008
• Agilent Hybridization oven and chambers
6
Multiplexing slides1x244K, 2x105K,
4x44K, 8x15K
• Bio-Rad Automated Electrophoresis system
Instrumentation 2009
7
Quantification of RNA
quality/concentration
RQI score for consistency
BIOPAD project (2005-2007)
• Microarray Capacity Building project– UP, Inqaba Biotec, WITS
• Focus areas– Training
• Workshops• Inqaba intern project
– Standard Operating Procedures– External support for projects
8
Workshops - BIOPAD
• Lab training (2005 & 2006)
9
Region Attendees
ACGT 22
Gauteng 10
SA 9
Workshops – Starter kit
10
Region Attendees
ACGT 9
Gauteng 4
SA 3
• Scaled down experiment (3 days)
Workshops
• ICTTD & Veterinary Science (2003)
11
Region Attendees
ACGT 12
Gauteng 3
SA 3
Outside SA 6
Workshops - Annual
• Data analysis– Genetics & Bioinformatics Hons– Interested researchers/students
• NBN– Data analysis for Bioinformatics students
12
Services – Project planning
• Experimental design
– Statistical relevance
• Choice of slides/platform
• Oligonucleotide design
13
Services – Sample QC
• RNA/DNA isolation• Experion
– RNA analyses – microarray, qPCR, RNAseq– DNA analyses – HTS
• Evaluation of external Bioanalyzer results
• Evaluation of RNA gels
14
Services – Hybridization
• Lab space and tech support
• Hyb-up chambers in waterbath– cDNA, oligo, gDNA
• Agilent hyb oven
15
Services – Scanning
• Spotted arrays – resolution 10 µm
• Agilent arrays – resolution 5 µm
• Single channel or dual channel scans
• Data backup at Facility
• Data capture assistance
16
Services – Data analysis
• Results file generation and QC
• Data normalization
• Statistical analysis + output
• Downstream protocols
• Annotation assistance
• GEO/ArrayExpress submission assistance
17
Software
• Spotverify– Verification of differential gene expression
• SSHscreen– SSH library quality evaluation
• SSH-DB– Moderated database for sequences, BLAST
• MADIBA– Annotation, biological function etc.
18
Communication
• Lectures to UP undergraduate students
• Lectures to UP postgraduate students
• Lectures to external undergraduate students
• Lectures to external postgraduate students
• Discussions and demos for visitors
19
Operating philosophy
• Non-profit
• No hybridisation service offered
– Student/Researcher training
– Increase capacity in techniques
– Make microarray technology accessible
20
Costing model
• Not full-cost
– Scientific Officer salary from UP
• Departmental responsibilities
• Reagents at cost price
• Data analysis with free software
– Scripts given to researchers for further use
21
Costing
22
Region Spotting (per slide)
Scanning(per slide)
Data analysis
ACGT R450 - R550 Free Free
Regional R450 - R550 R180 Free
External R600 – R750 R350 R350/h
Financial Administration
• Funds in UP cost point• Yearly financial statements to advisors• UP users:
– Internal fund transfers• External users
– Invoice issued by UP– Payments administered by UP Finance
• Excess funds for instrument maintenance
23
Projects by region
24
Region Past Current Future
ACGT 17 4 8
Gauteng 4 1 5
SA 4 1 0
Africa 1 0 2
Slides to date
25
Number
Slides printed 1970
Slides scanned 2261
Student training
Degree Number
Hons 3
MSc 11
PhD 17
Post-Doctoral 6
Principal Investigators 7
26
Publications
27
• 14 Publications in peer review journals
• 1 Book chapter
• 3 Hons reports
• 8 MSc theses
• 11 PhD theses
Challenges / Opportunities
• Spotter printing pins• Spotted arrays becoming obsolete• Scanner laser service• Scanner resolution• RNAseq
• Agilent slides becoming more accessible– Partnership with supplier – greater awareness
28
The Future
• New Plant Science building– Dedicated labs
• RNA isolation, Hybridization, Scanning
• Equipment needed– High resolution Agilent scanner
• 2 µm scanning resolution – high density• 1x1M, 2x400K, 4x180K, 8x60K
29
30
Thank you