Introduction to DNA microarrays DTU - January 2007 - Hanne Jarmer
Microarrays - The Concept Measure the level of transcript from a very large number of genes in one go CELL RNA
Why? RNA
How? gene m. RNA gene specific DNA probes labeled target
Microarrays - The Technologies Stanford-type Microarrays High-density
Stanford-type Microarrays
Stanford-type Microarrays Coating glass slides Deposition of probes Post-processing Hybridization
Spotting - Mechanical deposition of probes
16 -pin microarrayer
Microarrayer
Stanford microarrays CONTROL SAMPLE m. RNA c. DNA Cy 5 -c. DNA Cy 3 -c. DNA
® Gene. Chip Affymetrix oligonucleotide array • 11 to 20 oligonucleotide probes for each gene • On-chip synthesis of 25 mers • ~20, 000 genes per chip • good quality data • 280 -500 K features to play with
Photolithography in situ synthesis T A T T T A A T Mask #2 Mask #1 T A A T T T A AA A Spacers bound to surface with photolabile protection groups
Sample Preparation - Eberwine SAMPLE 42 °C 2 h + Reverse Transcriptase 16 °C 2 h RNA + RNase H + Polymerase 37 °C 6 h ss. DNA T 7 70 °C 10 min T 7 pol clean up ds. DNA + Biotin-labeled nucleotides a. RNA
Detection of Biotin (Affymetrix) Streptavidin Phycoerythrim = SAPE ( ) anti-SAPE Ig. G biotinylated anti-anti Ig. G
The Affymetrix Gene. Chip® A gene is represented like this: PM MM - Perfect Match (PM) - Mis. Match (MM) PM: CGATCAATTGCACTATGTCATTTCT MM: CGATCAATTGCAGTATGTCATTTCT
Nimble. Gen • 385, 000 to 2. 1 mill features • Long probes (up to 70 nt) • Service: -labelling -scanning -image analysis
Photolithography - Micromirrors
Tiling arrays are used for determation of genes, nc. RNAs, TF-binding sites, . . .
The DNA Array Analysis Pipeline Question Experimental Design Array design Probe design Sample Preparation Hybridization Buy Chip/Array Image analysis Normalization Expression Index Calculation Comparable Gene Expression Data Statistical Analysis Advanced Data Analysis Clustering PCA Classification Promoter Analysis Meta analysis Regulatory Network