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Utrecht Genomics Center
Microarray Facility
Joop van Helvoort Dec, 8th 2003
Frank Holstege Group
MicroarrayFacility
Bioinformatics TranscriptionRegulation
Dik van LeenenTony MilesMarian Groot KoerkampJoop van Helvoort
Utrecht Genomics Center (ABC/UMC)
Wijmenga Holstege Bos Burgering etc.
3.4 fte
Microarray facility: tasks
Microarray production
Protocol development
User support
User support: philosophy
Collaboration
As many groups as possible
Train users
User support: steps
1. Introductory discussions (1-2)goalsexperiment design experimental issues
2. Microarray course (1 week)practical
• performing a microarray experiment (wet lab)• image and data analysis (dry lab)
theory
3. Access to:arrayslab spaceequipmentsoftware
User support: arrays
DNA preprocessing – ready to spot
liquid handler
User support: arrays
100 m
150 m
Collection from 384 well plate
Deposition on glass surface
spotter
spotted material:
gene specific oligos (70-mer) or cDNAs
oligos for the external controls: for various purposes(van de Peppel et al., 2003, EMBO reports, 4, p.387-393)
suitable for two channel (Cy3 / Cy5) experiments;i.e. two sample comparison
User support: arrays
Human 1.1 array20 000 spots17 000 genes
3000 control spots
S. cerevisiae array16000 spots
6300 genes x23000 control spots
spots genes+splice variants
Human 1.1 20000 17000
Human 2.0 24500 21500
Yeast 16000 6300 (in duplo)
Rat 12000 4500 (in duplo)
coming soon:
Mouse 35000 32000
Dog (cDNA) 23000 20000
(collaboration with Jan Mol and Rene van den Ham, Fac. Vet. Med.)
All arrays contain 3000 control spots
Available Arrays
User support: lab space
4 benches available
User support: equipment
RNA/DNA quality and quantity assesment
Spectrophotometer: quantitycDNA synthesis (>30 µg total RNA)
RNA amplification (> 1 µg total RNA)
Total RNA
Bioanalyzer: integrity of RNA
(labeled) cDNA
User support: equipment
Microarray experiment itself:standard lab equipment
hybridisation:manually for nowautomated (future standard)
(Ventana)
Scanner (Agilent)
Image acquisition:state-of-the-art scanner
User support: equipment
Image analysis: transform tiff-image to data table Imagene 5.6
User support: equipment
Workstations
User support: equipment
Data quality check :• QQCC: QC
NormalizedRaw data
normalisation
etc.
User support: equipment
Data storage and advanced analysis :• Genespring 6.0 / Genet + BASE:
Financial Feasibility
Example:time course experiment: t = 0, 1, 2, 3, 4, 5 hourshuman samples, in duplorequires 10 microarrays
Microarray Affimetrix AgilentFacility
arrays 1250 8000 6000experiment 400 / 800 1000 1000support full no no
costs in euros
Costs: partial reimbursement
(for human: 1 microarray + 2 cDNA labeling + 1 hybridisation ~ 165 euro)
Current state of affairs
Funding:set up in 2000 with UMC financial support
and research grants Holstege + ABC funding since Oct. 2002
Users:29 users22 users within ABC
ContactContact
www.genomics.med.uu.nl
Joop van Helvoort Marian Groot Koerkamp
[email protected] [email protected]
Location: Stratenum