1 a decade of microarrays acgt microarray facility nicky olivier

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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

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People

• Director– Prof Dave Berger

• Scientific Officers– Danie Theron (2001-2006)– Nicky Olivier (2006-present)

• Statisticians– Loveness Dzikiti– Nanette Coetzer

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Advisors

Prof Jane Morris (ACGT)

Prof Henk Huismans (UP)

• Equipment and hardware acquisitions

• Approval of projects

• Approval Annual Financial Statements

• Future plans for Facility

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Start-up Instrumentation 2001

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Molecular Dynamics GenIII Spotter

Genepix 4000B Scanner532 nm / 635 nm

Instrumentation 2008

• Agilent Hybridization oven and chambers

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Multiplexing slides1x244K, 2x105K,

4x44K, 8x15K

• Bio-Rad Automated Electrophoresis system

Instrumentation 2009

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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

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Workshops - BIOPAD

• Lab training (2005 & 2006)

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Region Attendees

ACGT 22

Gauteng 10

SA 9

Workshops – Starter kit

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Region Attendees

ACGT 9

Gauteng 4

SA 3

• Scaled down experiment (3 days)

Workshops

• ICTTD & Veterinary Science (2003)

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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

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Services – Project planning

• Experimental design

– Statistical relevance

• Choice of slides/platform

• Oligonucleotide design

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Services – Sample QC

• RNA/DNA isolation• Experion

– RNA analyses – microarray, qPCR, RNAseq– DNA analyses – HTS

• Evaluation of external Bioanalyzer results

• Evaluation of RNA gels

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Services – Hybridization

• Lab space and tech support

• Hyb-up chambers in waterbath– cDNA, oligo, gDNA

• Agilent hyb oven

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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

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Services – Data analysis

• Results file generation and QC

• Data normalization

• Statistical analysis + output

• Downstream protocols

• Annotation assistance

• GEO/ArrayExpress submission assistance

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Software

• Spotverify– Verification of differential gene expression

• SSHscreen– SSH library quality evaluation

• SSH-DB– Moderated database for sequences, BLAST

• MADIBA– Annotation, biological function etc.

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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

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Operating philosophy

• Non-profit

• No hybridisation service offered

– Student/Researcher training

– Increase capacity in techniques

– Make microarray technology accessible

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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

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Costing

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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

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Projects by region

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Region Past Current Future

ACGT 17 4 8

Gauteng 4 1 5

SA 4 1 0

Africa 1 0 2

Slides to date

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Number

Slides printed 1970

Slides scanned 2261

Student training

Degree Number

Hons 3

MSc 11

PhD 17

Post-Doctoral 6

Principal Investigators 7

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Publications

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• 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

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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

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Thank you