Liver samples were extracted from each mouse as described previously (Shockley and Churchill, 2006). Total RNA was reverse transcribed with oligo(dT)-T7 primers. before double-stranded cDNA was generated with the Superscript double-stranded cDNA synthesis custom kit (Invitrogen).
Label
biotin
Label protocol
The cDNA was linearly amplified with biotinylated nucleotides (Enzo Diagnostics, Farmingdale, NY) through an in vitro transcription reaction with T7 RNA polymerase.
Hybridization protocol
Fifteen micrograms of biotin-labeled and fragmented cRNA was then hybridized onto MOE430v2.0 GeneChip® arrays for 16 hours at 45°C. Post-hybridization staining and washing were performed according to the manufacturer’s protocols using the Fluidics Station 450 instrument (Affymetrix, Santa Clara, CA).
Scan protocol
Arrays were scanned with a GeneChip® Scanner 3000 laser confocal slide scanner. Images were quantified using GeneChip Operating Software (GCOS) v1.2 (Affymetrix).
Description
One group of mice was fed an atherogenic high-fat (30% fat) diet containing cholic acid to increase fat uptake and another was fed a low-fat (6% fat) regular chow diet. Males and females from both diets were studied for mouse strains 129S1/SvImJ, A/J, BALB/cJ, C3H/HeJ, C57BL/6J, CAST/EiJ, DBA/2J, I/LnJ, MRL/MpJ-Tnfrsf6lpr/J, NZB/BINJ, PERA/Ei, and SM/J. All strains were sacrificed between 11- and 13 weeks of age except for CAST and PERA, which were harvested after 50 weeks of age. CAST and PERA were subsequently removed from our analysis based on discrepant harvest age, but can be found in our database (see below). Three replicate animals were used for each combination of diet, strain, and sex, resulting in a total of 120 mice surveyed for gene expression.
Data processing
RMA values are in the VALUE column here. Briefly, the RMA method was used to adjust the background of perfect match (PM) probes, apply a quantile normalization of the corrected PM values and calculate final expression measures using the Tukey median polish algorithm.
