Microarrays: Tools & Interpretation of Data

1. Microarrays:Tools & Interpretation of Data Kirsty Jensen

2. Microarrays • What is a microarray? • How the technology has evolved • Macroarrays • Glass microarrays • Affymetrix • Designing a microarray experiment • Choice of arrays • Experimental design • RNA samples • Analysis • Scanning • Quantification of spots • Normalization • Statistical analysis • Identification of genes of interest • Validation

3. What Is A Microarray? “A microarray is a tool for analyzing gene expression that consists of a small membrane or glass slide containing samples of many genes arranged in a regular pattern.”

4. Uses • Analysis of gene expression • SNP analysis • MHC typing • Identification of pathogens • Identification of tumours

5. Microarrays • The underlying principle of microarray technology is the ability of DNA to bind to itself and to RNA. • Analyzing gene expression involves the detection of mRNA species (transcriptome) present in a cell or tissue at a particular point in time.

6. Evolution of Microarrays • One gene / one experiment • Northern blots, RNase assays, RT-PCR • Hypothesis driven • Macroarrays • Nylon membranes • 32P labelled probes

7. RNA RNA 32P labelled cDNA 32P labelled cDNA Hybridization Hybridization Macroarrays

8. Evolution of Microarrays • One gene / one experiment • RT-PCR, northern blots, RNase assays • Hypothesis driven • Macroarrays • Nylon membranes • 32P labelled samples • Microarrays • Glass slides • Fluorescent labelled samples • Two samples per microarray

9. RNA RNA Cy3 labelled cDNA Cy5 labelled cDNA Hybridization Microarrays

10. Glass Microarrays • cDNA arrays • >1000 nucleotides in length • cDNA libraries • Oligonucleotide arrays • 70-80 nucleotides in length • Increased specificity

11. Evolution of Microarrays • One gene / one experiment • RT-PCR, northern blots, RNase assays • Hypothesis driven • Macroarrays • Nylon membranes • 32P labelled samples • Microarrays • Glass slides • Fluorescent labelled samples • Affymetrix

12. Affymetrix • 25 nucleotides in length • Mismatch pair • Single mismatch at centre of probe • Used to detect & eliminate false results • 11 probe pairs per gene • Photolithography

13. Affymetrix - Construction

14. Affymetrix

15. RNA cDNA Biotin-cRNA Hybridization Streptavidin-phycoerythrin Affymetrix

16. Bovine Microarrays

17. Conclusions (1) • Microarrays have many uses. • Major use is to investigate gene expression. • mRNA isolated from a cell gives a snap-shot of the events occurring at that time. • There are 3 main types of microarrays • Glass cDNA microarrays • Glass oligonucleotide microarrays • Affymetrix