User meeting ABC/UMC Microarray Technology Group, March 2004

1. User meeting ABC/UMC Microarray Technology Group, March 2004

2. Genomics Center Utrecht (ABC/UMC) Wijmenga Holstege Voest Bos etc. Microarray Technology Group Bioinformatics Transcription Regulation Dik van Leenen Tony Miles Marian Groot Koerkamp Joop van Helvoort 3.4 fte

3. Program Introduction: Goals How the microarray facility works Guidelines Example studies Wet lab work (Joop van Helvoort)

4. 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

5. Many users Encourage: move wet lab work to own lab communicate by email: genomics-array@med.uu.nl stick to protocols use information on website: http://www.microarrays.med.uu.nl follow lab rules (and ask if in doubt)

6. Goal Make microarray technology available in Utrecht so that many groups can publish high quality studies Are (technically) high quality studies possible?

7. 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

8. etc. SRB2 deletion MED3 deletion WT controls Array#1 mt1 cy5 wt ref cy3 Array#2 wt ref cy5 mt2 cy3 Array#1 mt1 cy5 wt ref cy3 Array#2 wt ref cy5 mt2 cy3 Array#1 wt1 cy5 wt ref cy3 Array#2 wt ref cy5 wt2 cy3 16 mutants 6 wt controls Select for genes significantly changed in any deletion but not in same vs same, wt controls 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

9. 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

10. Such comparative studies are not restricted to yeast Analysis of head-neck tumors (in collaboration with dept. Pathology)

11. 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

12. _ ... and collaborations Srb/Mediator Jeroen van de Peppel Nienke Kettelarij HNSCC Paul Roepman Piet Slootweg (Pathology) Marcel Tilanus (Pathology) Microarray technology Dik van Leenen Tony Miles Marian Groot-Koerkamp Joop van Helvoort