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Central Dogma 2

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

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Central Dogma 2

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

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

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

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

  6. leukemia1

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

  8. DNA- DNA hybridization1 RNA- DNA uracil

  9. kT MISMATCH kT kT kT 7-17 PERFECT MATCH hybridization2

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

  11. cDNA spots

  12. B B B B B B B B B B B B Affymetrix Experimental Design: Target Biotin - labeled cRNA transcript Cells Poly (A)+ RNA Or Total RNA IVT Biotin-UTP Biotin-CTP Fragment heat, Mg2+ cDNA Hybridize (16 hours) Wash & Stain Biotin - labeled cRNA fragments Scan (75 minutes) (8 minutes)

  13. Affymetrix overview: Probe Array

  14. Probe preparation: Photolithographic synthesis Lamp Mask Chip

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

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

  17. B B B B B B B B B B B B Affymetrix Experimental Design Biotin - labeled cRNA transcript Cells Poly (A)+ RNA Or Total RNA IVT Biotin-UTP Biotin-CTP Fragment heat, Mg2+ cDNA Hybridize (16 hours) Wash & Stain Biotin - labeled cRNA fragments Scan (75 minutes) (8 minutes)

  18. B B B B B B B B B B B B Affymetrix Experimental Design Biotin - labeled cRNA transcript Cells Poly (A)+ RNA Or Total RNA IVT Biotin-UTP Biotin-CTP Fragment heat, Mg2+ cDNA Hybridize (16 hours) Wash & Stain Biotin - labeled cRNA fragments Scan (75 minutes) (8 minutes)

  19. RT + labeling

  20. 4F5-74 REVERSE TRANSCRIPTASE

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

  22. RT + labeling

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

  24. Photolithographic Synthesis Lamp Mask Chip

  25. Synthesis of Ordered Oligonucleotide Arrays Light (deprotection) Mask TTOOO OOOOO HO HO O O O T – Substrate Light (deprotection) Mask CATAT AGCTG TTCCO TTCCG TTOOO C – Substrate REPEAT

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

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

  28. Shirley’s Affy incubator

  29. Shirley’s Affy scanner

  30. pixels

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

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

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

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

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

  37. Experiment Differential expression Different RNAs

  38. end

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