Target was prepared and hybridized according to the Affymetrix Technical Manual. Total RNA (10 ug) was converted into cDNA using Reverse Transcriptase (Invitrogen) and a modified oligo(dT)24 primer that contains T7 promoter sequences (GenSet).
Hybridization protocol
The cDNA products were incubated with T7 RNA Polymerase and biotinylated ribonucleotides using an In Vitro Transcription kit (Affymetrix). The resultant cRNA product was purified using an RNeasy column (Qiagen) and quantified with a spectrophotometer. The cRNA target (20ug) was incubated at 94ºC for 35 minutes in fragmentation buffer (Tris, MgOAc, KOAc). The fragmented cRNA was diluted in hybridization buffer (MES, NaCl, EDTA, Tween 20, Herring Sperm DNA, Acetylated BSA) containing biotin-labeled OligoB2 and Eukaryotic Hybridization Controls (Affymetrix). The hybridization cocktail was denatured at 99°C for 5 minutes, incubated at 45°C for 5 minutes and then injected into a GeneChip cartridge. The GeneChip array was incubated at 42°C for at least 16 hours in a rotating oven at 60 rpm. GeneChips were washed with a series of nonstringent (25°C) and stringent (50°C) solutions containing variable amounts of MES, Tween20 and SSPE. The microarrays were then stained with Streptavidin Phycoerythrin and the fluorescent signal was amplified using a biotinylated antibody solution.
Scan protocol
Fluorescent images were detected in an GeneChip® Scanner 3000
Description
Control dataset for subsequent analysis of drug responses
Data processing
Expression data was extracted using the GeneChip Operating System v 1.1 (Affymetrix). All GeneChips were scaled to a median intensity setting of 500.
