1. Microarray (Gene Chip) Technology

2. PART 1 Part 2 Part 3 Part 4

5. Features – Parallelism • Thousands of genes simultaneously – Miniaturization • Small chip size – Multiplexing • Multiple samples at the same time – Automation • Chip manufacturing • Reagents

15. Part 1 PART 2 Part 3 Part 4

22. Step Step Step

23. Step 2 Step 1 Step 3 Step 2 Step 3

31. Microarray Fabrication • Fabrication via Printing (cDNA chip) • DNA sequence stuck to glass substrate • DNA solution pre-synthesized in the lab • Fabrication In Situ(Affymetrix chip) • Sequence “built” • Photolithographic techniques use light to release capping chemicals • 365 nm light allows 20-m resolution

32. Fabrication via printing cDNA chip Fabrication In Situ: Affymetrix chip

33. Microarray (Gene Chip) Technology

35. Microarray Features – Parallelism • Thousands of genes simultaneously – Miniaturization • Small chip size – Multiplexing • Multiple samples at the same time – Automation • Chip manufacturing • Reagents

37. Afftmetrix chip

39. cDNA chip

40. cDNA chip

41. Fabrication via printing cDNA chip Fabrication In Situ: Affymetrix chip

42. Microarray experiment and data acquisition

43. Poly-A mRNA 10% Biotin-labeled Uracil Antisense cRNA cDNA IVT AAAA Cells L L L L L L Fragment (heat, Mg2+) Labeled fragments Hybridize Wash/stain Scan Affymetrix microarray process

44. L L L L L L L L L L L L Hybridization and Staining Biotin Labeled cRNA GeneChip Hybridized Array + + SAPE Streptavidin- phycoerythrin

45. The Result • A light source scans the array, causing the dyes to fluoresce • The glow is picked up by a sensor and is used to determine the relative abundance of the RNA • This information must be processed to determine the level of activity for each expressed gene