1. Analysis of Microarray Data 1.Scan the images 2.Quantify intensity of spots 3.Normalization 4.Analysis of data 5.Identification of genes of interest 6.Validation

2. Analysis of Microarray Data 1.Scan the images 2.Quantify intensity of spots 3.Normalization 4.Analysis of data 5.Identification of genes of interest 6.Validation

3. Analysis of Microarray Data 1.Scan the images 2.Quantify intensity of spots 3.Normalization 4.Analysis of data 5.Identification of genes of interest 6.Validation

4. Quantification of Spot Intensity BlueFuse QuantArray ScanAlyze NHGRI – Scanalytics Imagene GenePix ArrayVision TIGR Spotfinder

5. BlueFuse

6. Analysis of Microarray Data 1.Scan the images 2.Quantify intensity of spots 3.Normalization 4.Analysis of data 5.Identification of genes of interest 6.Validation

7. Normalization To identify and remove sources of systematic variation in the measured fluorescence intensities due to factors other than differential expression: –Spatial effects –Dye effects Comparison of expression levels within and between microarrays.

8. Spatial Effects

9. MA Plots Before normalization After normalization M = log 2 R – log 2 G A = (log 2 R+ log 2 G)/2

10. Analysis of Microarray Data 1.Scan the images 2.Quantify intensity of spots 3.Normalization 4.Analysis of data 5.Identification of genes of interest 6.Validation

11. Analysis of Data GeneSpring

12. Analysis of Microarray Data 1.Scan the images 2.Quantify intensity of spots 3.Normalization 4.Analysis of data 5.Identification of genes of interest 6.Validation

13. Identification of Genes of Interest Microarray analysis can identify hundreds of differentially expressed genes. How do you identify genes of particular interest?

14. Data Mining Tools Literature searches –Chilibot (www.chilibot.net)www.chilibot.net –Protein Annotator’s Assistant (www.ebi.ac.uk/paa)www.ebi.ac.uk/paa –iHOP (www.pdg.cnb.uam.es/UniPub/iHOP)www.pdg.cnb.uam.es/UniPub/iHOP Pathway searches –DAVID & EASE (www.david.niaid.nih.gov/david)www.david.niaid.nih.gov/david –Biorag (www.biorag.org)www.biorag.org Transcription factor binding sites –MatInspector (www.genomatrix.de)www.genomatrix.de

15. Analysis of Microarray Data 1.Scan the images 2.Quantify intensity of spots 3.Normalization 4.Analysis of data 5.Identification of genes of interest 6.Validation

16. Validation Confirm microarray results by: –qRT-PCR –Northern blots Confirm biological significance by investigating protein expression: –Western blots

17. Conclusions (3) Several stages are involved in analyzing microarray data: –Scan images –Quantify spots –Statistical analysis There are many different soft-ware packages and methods for this analysis. Further analysis is required to pin-point genes of interest. Microarray results need to be validated by other methods.

18. Conclusions (general) Microarray experiments are expensive. Microarray experiments are time-consuming. Microarray experiments generate huge amounts of data. The quality of the data obtained from a microarray experiment depends on: –Design of the experiment –Quality of RNA –Statistical analysis –Interpretation of the data There is, at present, no definitive method for either designing or analyzing a microarray experiment.