The dissected mice livers were immediately stored in RNA later (Ambion). Total RNA was extracted with Trizol reagent according to the manufacturer’s instructions (Invitrogen), except that the precipitated RNA pellet was washed in 80% ethanol and not vortexed.
Label
biotin
Label protocol
Total RNA was processed following the GeneChip Expression 30-Amplification Reagents One-cycle cDNA synthesis kit instructions (Affymetrix Inc, Santa Clara, CA, USA) to produce double-stranded cDNA. This was used as a template to generate biotin-labelled cRNA following manufacturer’s specifications.
Hybridization protocol
Fifteen micrograms of biotin-labelled cRNA was fragmented and 10 mg were hybridized onto Affymetrix Mouse Genome 430 2.0 arrays overnight in the GeneChip Hybridisation oven 6400 using standard procedures, The arrays were washed and stained in a GeneChip Fluidics Station 450.
Scan protocol
Scanning was carried out using the GeneChip Scanner 3000 and image analysis was performed using GeneChip Operating Software.
