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

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

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)

excel

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

leukemia1

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

hybridization1 DNA- DNA RNA- DNA uracil

hybridization PERFECT MATCH MISMATCH kT

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

cDNA spots

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)

Affymetrix overview: Probe Array

Probe preparation: Photolithographic synthesis Lamp MaskChip

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

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

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)

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)

RT + labeling

4F5-74 REVERSE TRANSCRIPTASE

3F6-82 retrovirus life cycle 3F6-82

RT + labeling

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

Photolithographic Synthesis Lamp MaskChip

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

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

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

Shirley’s Affy incubator

Shirley’s Affy scanner

pixels

excel

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

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

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

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

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

Experiment Different RNAs Differential expression

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