1. Parallel human genome analysis: Microarray-based expression monitoring of 1000 genes Mark Schena, Dari Shalon, Renu Heller, Andrew Chai, Patrick O. Brown, and Ronald W. Davis

2. Schena et. al • Goals • To detect the expression of thousands of genes simultaneously • Gene expression studies – expression patterns of genes provide clues to function by comparison • Gene discovery studies • Use the microarray assay to identify • Known and novel heat shock genes • Phorbol-ester regulated genes

3. Schena et. al Types of Microarrays • Photolithography – using oligos • Spotting – using cDNAs/ESTs

4. Schena et. al Methods • Human cDNA from human T mRNA transformed by the Epstein Barr Virus with 5’ amino acid modification, amplified by PCR, and arrayed onto silyated microscope slides • Probes labeled with fluorescin and Cy5-dCTP are hybridized to 1056-element array and scanned • Verify expression patterns with RNA Blot • Array elements that display differential expression patterns are sequenced • Compare sequence to Informatics databases

5. Schena et. al, Figure 1 Monitoring of heat shock response in control (37o)and treated Jurkat (43o,human T) cells - Array contains 10 Arabidopsis controls, 1046 human blood cDNAs • -White box indicates altered fluorescence: • Red boxes indicate activation • Green boxes indicate repression Hybridization signals observed for > 95% of human cDNA elements Comparative expression finds altered fluorescence in 17 array elements

6. Schena et. al, Figure 2 Elemental displays of activated and repressed genes • Fluorescin labeled probes from (+) heat-shock and (+) phobol ester cells are compared to Cy5-labeled untreated probes • Data is the average of the ratios from the 2 hybridizations • 17 elements have a 2-fold alteration in fluorescence • Intensity of fluorescnece is a measure of mRNA abundance

7. Schena et. al, Table 1 • 14/17 clones matched; proximal and distal ends map to same gene • Hsp90, dnaJ, polyubiquitin, tcp-1 are highly induced • Novel sequences (B7-B9) have 2-fold induction

8. Schena et. al, Table 2 Correlation of human gene expression from microarray analysis is confirmed by RNA blot analyses

9. Schena et. al, Figure 3 Microarrays measureexpression in human tissuesBone marrow, brain, prostate, and heart Expression levels in genes correlates with expression level in tissues

10. Advantages Schena et. al • Small hybridization volumes using cDNA provides specificity not possible with oligo-based arrays • High array densities • Incorporation of fluorescence labeling and detection • High throughput: sequence-based methods require serial processing • Rich number of ESTs makes for more powerful arrays

11. Schena et. al Disadvantages • Cost • Commercial availability of microarrays

12. Conclusions Schena et. al • Microarrays are useful for gene discovery in the absence of sequence information • Parallel assays can monitor gene expression for thousands of genes • Allows high throughput human genome expression and gene discovery • Allows for rapid mechanistic examination of hormones, drugs, elicitors, and other small molecules • Potential capacities for patient screening • Sensitivities of microarrays allows for functional analysis of transcription factors, kinases, growth factors