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User meeting ABC/UMC Microarray
Technology Group, March 2004
Genomics Center Utrecht (ABC/UMC)
Wijmenga
Microarray
Technology Group
Dik van Leenen
Tony Miles
Marian Groot Koerkamp
Joop van Helvoort
Holstege
Bioinformatics
3.4 fte
Voest
Transcription
Regulation
Bos
etc.
Program
Introduction:
Goals
How the microarray facility works
Guidelines
Example studies
Wet lab work (Joop van Helvoort)
Goal
Make microarray technology available in Utrecht so that
many groups can publish high quality studies
Consequences
house facility within a research group that uses technology
open facility
DIY: using our arrays, expertise and equipment
Expect
collaborative attitude
partial cost recovery (<15%)
co-authorship
Many users
Encourage:
move wet lab work to own lab
communicate by email: [email protected]
stick to protocols
use information on website: http://www.microarrays.med.uu.nl
follow lab rules
(and ask if in doubt)
Goal
Make microarray technology available in Utrecht so that
many groups can publish high quality studies
Are (technically) high quality studies possible?
Comparing functionally related mutants
Srb/mediator complex (conserved yeast – mammals)
24 subunits
associated with RNA polymerase II
involved in regulation of transcription
Role of different subunits in yeast?
CDK-Cyclin
Tail
Head
Middle
Experiment design
All hybridisations with common wild-type reference RNA
Each deletion strain grown in duplicate
- dye swap on two arrays
- genes in duplicate: 4 data points per gene per deletion
Additional wild-type controls grown alongside each deletion strain
- biological and technical variation
SRB2 deletion
MED3 deletion
Array#1
Array#2
mt1 cy5
wt ref cy5
wt ref cy3 mt2 cy3
Array#1
Array#2
mt1 cy5
wt ref cy5
wt ref cy3 mt2 cy3
16 mutants
etc.
WT controls
Array#1
Array#2
wt1 cy5
wt ref cy5
wt ref cy3 wt2 cy3
6 wt controls
Select for genes significantly changed in any deletion but not in
same vs same, wt controls
Are (technically) high quality studies possible?
Yes! (but requires discipline)
Rejected experiments:
Total RNA and mRNA isolation deviated
Label incorporation deviated
Hybridisations heterogeneous, weak or too much background
Parallel wt controls not similar
Chromosomal abberations
Duplo’s not similar
Not strictly necessary for all microarray experiments
(e.g. screens)
But still recommended
Such comparative studies are not
restricted to yeast
Analysis of head-neck tumors
(in collaboration with dept. Pathology)
Comparative studies (and screens)
Require strict attention to experimental detail
Standardized arrays and protocols
Controls to generate estimates of variation
Enough data per gene/sample, including dye swap
QC of RNA, labeled material and hybs
Also requires analysis tools
and collaborations
Srb/Mediator
Jeroen van de Peppel
Nienke Kettelarij
_
HNSCC
Paul Roepman
Piet Slootweg (Pathology)
Marcel Tilanus (Pathology)
Exit from stationary phase
SP
15 60 180
360 min
Microarray technology
Dik van Leenen
Tony Miles
Marian Groot-Koerkamp
Joop van Helvoort
Genes
SP
15 60 180
360 min
...
External controls & cell count