The RNA was extracted using RNAqueous Kit (Ambion) as per the instructions of the manufacturer. Briefly, media was removed from the plates and cells were lysed in 1 ml lysis buffer. To this was added 1 ml of 64% alcohol. The contents were mixed and passed through the column. The RNA bound to the column was washed once with wash solution 1 and twice with wash solution 2/3. Finally RNA was eluted in 40 ul of hot elution buffer. RNA was quantified by spectrophotometry and quality verified by running 5 ug of RNA on 1% agarose gel. The resulting RNA was used for microarray analyses.
Label
biotin-cRNA, streptavidin-phycoerythrin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
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 min, incubated at 45°C for 5 min and then injected into a GeneChip cartridge. The GeneChip array was incubated at 42°C for at least 16 hr 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 a GeneChip® Scanner 3000.
Description
25W_A
Data processing
Expression data were extracted using the MicroArray Suite 5.0 software (Affymetrix). All GeneChips were scaled to a median intensity setting of 500.
