1. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Richard Talbot Roslin Institute and R(D)SVS University of Edinburgh Microarrays overview, one and two colour systems

2. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Microarray uses Definition A microarray is a high density of DNA probes of known sequence arrayed across a solid surface Uses Gene Expression Comparative Genome Hybridisation (CGH) Single Nucleotide polymorphisms (SNP) DNA methylation Copy Number variation (CNV)

3. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Microarray experiment Define the questions Choose microarray RNA preparation Target labelling Array Hybridisation Image scanning and analysis Statistical analysis Reporting results

4. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Experimental model Gene expression in the developing limb model: During development different genes are expressed in a choreographed order for the spatial patterning of tissues. Chick limb buds at stage 20 (~3 days) show patterning of Sonic Hedgehog a major transcription factor controlling the anterior-posterior axis in the limb bud.

5. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Experimental questions 1.What are the genes associated with patterning in the limb bud? 2.What are the genes influenced by the presence of Sonic Hedgehog protein?

6. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Experimental plans Posterior expression Anterior expression Question 1 V’s Anterior bead expression Posterior expression V’s Anterior expression Anterior Bead expression V’s Question 2 Anterior Gli3 expression Posterior expression V’s Anterior expression Anterior GLi3 expression V’s

7. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Reference design Reference Anterior bead expression Anterior expression Posterior expression Anterior Gli3 expression One Colour Experiment

8. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Microarray experiment Define the questions Choose microarray RNA preparation Target labelling Array Hybridisation Image scanning and analysis Statistical analysis Reporting results

9. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Types of Microarrays Probe type cDNA Oligonucleotide short (<35 bases) Oligonucleotide long (>35 bases)

10. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Oligonucleotide synthesis

11. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Microarray experiment Define the questions Choose microarray RNA preparation Target labelling Array Hybridisation Image scanning and analysis Statistical analysis Reporting results

12. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals RNA quality

13. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Microarray experiment Define the questions Choose microarray RNA preparation Target labelling Array Hybridisation Image scanning and analysis Statistical analysis Reporting results

14. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals RNA Labelling Total RNA cDNA Reverse transcription AAAAAA Aminoallyl-UTP aaaa aaaa aaaa aaaa cDNA **** **** **** **** Cy3*-CTP or Cy5*-CTP **** **** **** **** Dye Coupling Cy3* or Cy5* IndirectDirect

15. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals RNA amplification Total RNAcDNA Reverse transcription In Vitro transcription Remove RNA and ds-cDNA AAAAAA AAAAAA-T7 TTTTTT-T7 AAAAAA-T7 TTTTTT-T7 AAAAAA-T7 TTTTTT-T7 AAAAAA-T7 TTTTTT-T7 T7 cRNA

16. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Affymetrix Single Channel Microarray Experiment

17. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Microarray experiment Define the questions Choose microarray RNA preparation Target labelling Array Hybridisation Image scanning and analysis Statistical analysis Reporting results

18. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Microarray Experiment Reverse Transcription Dye Coupling Cy3 Cy5 Mix on Slide Excitation Emission Hybridise Scanning Control RNATreatment RNA

19. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Hybridisation Temperature control Mixing Multi-chamber Loading sample Washing

20. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Scanning Set laser power Set PMT gain or auto adjust Set scan area Apply griding file Align griding file Flag problem features Extract Values

21. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Microarray experiment Define the questions Choose microarray RNA preparation Target labelling Array Hybridisation Image scanning and analysis Statistical analysis Reporting results

22. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Sources of variation 1 Microarray production Print-pin anomalies Variations in printed probe quantities even with the same pin Chip batch variation (due to many sources of unknown variations) Differences in sequence length of the immobilized DNA Variations in chemical probe attachment levels to the slide mRNA Differences in conditions. Differences between experimental subjects within the same covariate level. Differences between samples from the same subject. Variation in mRNA extraction methods from original sample. Variations in reverse transcription. Differences in PCR amplification. Different labelling efficiencies.

23. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Sources of variation 2 Scanning/imaging Different scanners. Different photo-multiplier gain. Different spot-finding software. Different grid alignments. Different laser power Hybridization process Different dye sensitivities. Inequalities in the application of mRNA to the slide. Variations in the washing efficiencies of non-hybridized mRNA on the slide. Other differences in hybridization parameters, such as: temperature experimenter time of the day

24. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Normalisation (two dye) Used to remove systematic bias form the image data Methods Global All genes Housekeeping/spike genes Loess Global Pin tip Spatial

25. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Normalisation (one dye) Method depends on slide system Affymetrix MAS5.0 RMA Plier Agilent Spatial normalisation

26. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Differential expression T-Test Multiple testing - False Discovery Rate (FDR) ANOVA

27. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Microarray experiment Define the questions Choose microarray RNA preparation Target labelling Array Hybridisation Image scanning and analysis Statistical analysis Reporting results

28. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals M inimal I nformation A bout a M icroarray E xperiment Database of microarray experiment Samples Data Analysis Protocols Experiment ARRAY Hybridisation

29. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Gene Expression Data Repositories Major public repositories ArrayExpress (EBI) - http://www.ebi.ac.uk/microarray-as/ae/ GEO (NCBI) - http://www.ncbi.nlm.nih.gov/geo/ CIBEX (DDBJ) – http://cibex.nig.ac.jp/index.jsp Submission tools Tab2Mage - http://www.ebi.ac.uk/cgi-bin/microarray/tab2mage.cgi GEOarchive - http://www.ncbi.nlm.nih.gov/projects/geo/info/spreadsheet.html#G EOarchive Cibex - http://cibex.nig.ac.jp/submission/index.jsp

30. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals After the Gene list Electronic Annotation Clustering Pathways Text mining Networks Practical qPCR In Situ hybridisation Northern blot Western Blot Immunohistochemistry siRNA Pharmacology

31. ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals Conclusions Several successful microarray platforms are available Choice of platform is based on experimental design and cost It is key to know the questions being asked Many steps in a microarray experiment contribute to the overall variance Normalisation can be used to remove technical variance Microarrays are a tool to answer a specific question have a plan on how to confirm the answer.