1. Central Dogma 2 Transcription mRNA Information stored In Gene (DNA) Translation Protein Transcription Reverse Transcription SELF-REPAIRING ARABIDOPSIS, Nature March 05, NY Times (R Pruitt) Storage in RNA??? Cells express different subset of the genes In different tissues and under different conditions

2. REGULATION AT DIFFERENT STEPS control M-RNA IS PROCESSED (SPLICED) TRANSCRIPTIONAL CONTROL RNA PROCESSING CONTROL RNA TRANSPORT CONTROL RNA DEGRADATION PROTEIN ACTIVITY TRANSLATIONAL CONTROL

3. MEASURING GENE EXPRESSION PROFILE WHEN A PARTICULAR GENE IS EXPRESSED, THE CONCENTRATIONS OF ITS CORRESPONDING MESSENGER RNA AND PROTEIN ARE HIGH. Measure Gene expression A DNA-CHIP MEASURES CONCENTRATIONS OF THOUSANDS OF DIFFERENT MESSENGER RNA LATEST AFFYCHIP: U133P2 – 54,675 (probesets)

4. excel

5. Low High VISUALIZATION: HOW DOES ONE SHOW SO MANY NUMBERS? COLOR CODE: REPRESENT NUMBERS BY COLORS Color code

6. leukemia1

7. gene 1000 Sample # 57 LEUKEMIA DATA (Rozovskaia, Canaani) E ij = EXPRESSION LEVEL OF GENE i IN SAMPLE j Leukemia2

8. hybridization1 DNA- DNA RNA- DNA uracil

9. hybridization2 7-17 PERFECT MATCH MISMATCH kT

10. cDNA microarray expt (M.Campbell movie) cDNA SPOTTED ARRAYS: QUICK OVERVIEW

11. cDNA spots

12. Affymetrix Experimental Design: Target cDNA Fragment heat, Mg 2+ Wash & Stain Scan Hybridize (16 hours) Biotin - labeled cRNA transcript Poly (A) + RNA Or Total RNA IVT Biotin-UTP Biotin-CTP Biotin - labeled cRNA fragments Cells B B BB B B B B B B B B (8 minutes) (75 minutes)

13. Affymetrix overview: Probe Array

14. Probe preparation: Photolithographic synthesis Lamp MaskChip

15. Wafer and Chip Format 1.28cm 11 µm Millions of identical oligonucleotide probes per feature (25 base-long single - strand DNA) 49 - 400 chips/wafer up to ~ 1,200,000 features/chip

16. Perfect Match/MisMatch U133Plus2 – 11 probe pairs / probe set

17. Affymetrix Experimental Design cDNA Fragment heat, Mg 2+ Wash & Stain Scan Hybridize (16 hours) Biotin - labeled cRNA transcript Poly (A) + RNA Or Total RNA IVT Biotin-UTP Biotin-CTP Biotin - labeled cRNA fragments Cells B B BB B B B B B B B B (8 minutes) (75 minutes)

18. Affymetrix Experimental Design cDNA Fragment heat, Mg 2+ Wash & Stain Scan Hybridize (16 hours) Biotin - labeled cRNA transcript Poly (A) + RNA Or Total RNA IVT Biotin-UTP Biotin-CTP Biotin - labeled cRNA fragments Cells B B BB B B B B B B B B (8 minutes) (75 minutes)

19. RT + labeling

20. 4F5-74 REVERSE TRANSCRIPTASE

21. 3F6-82 retrovirus life cycle 3F6-82

22. RT + labeling

23. may.ppt Wafer and Chip Format 1.28cm 20 - 50 µm Millions of identical oligonucleotide probes per feature 49 - 400 chips/wafer up to ~ 400,000 features/chip

24. Photolithographic Synthesis Lamp MaskChip

25. Synthesis of Ordered Oligonucleotide Arrays O O O O OO O O O O Light (deprotection) HO HO O O O T T O O OT T O O O T T C C OT T C C O Light (deprotection) T T O O OT T O O O C A T A TC A T A T A G C T GA G C T G T T C C GT T C C G Mask Substrate Mask Substrate T – C – REPEAT

26. Perfect Match/MisMatch U133Plus2 – 11 probe pairs / probe set

27. Hybridization and Staining Array cRNA Target Hybridized Array Streptravidin- phycoerythrin conjugate

28. Shirley’s Affy incubator

29. Shirley’s Affy scanner

30. pixels

31. excel

32. Replicates by Scatter-Plot cells RNA cRNA hyb. cocktail chip scan analyze

33. Technical Replicate I cells RNA cRNA hyb. cocktail chip scan analyze scan analyze Same array, 2 scans (diff. scanners, days)

34. Technical Replicate II cells RNA cRNA hyb. cocktail chip scan analyze chip scan analyze Same cRNA, 2 arrays (diff. days)

35. Technical Replicate III cells RNA cRNA hyb. cocktail chip scan analyze cRNA hyb. cocktail chip scan analyze Same RNA, 2 preps

36. Biological Replicate cells RNA cRNA hyb. cocktail chip scan analyze cells RNA cRNA hyb. cocktail chip scan analyze 2 RNAs, 2 preps

37. Experiment Different RNAs Differential expression

38. end