Extraction using TRIZOL following manufacturer's protocol
For each array, up to 10 mg of total RNA were converted to cDNA. Biotinylated cRNA was then produced in vitro using the GeneChip expression 3′ amplification one-cycle target labeling kit (Affymetrix)
Microarrays were hybridized with fragmented biotinylated cRNA for 16 h at 45°C with constant rotation (45 r.p.m.), and processed using the Affymetrix GeneChip Fluidic Station 450. Streptavidin-conjugated phycoerythrin (SAPE) was used for staining, followed by amplification using a biotinylated anti-streptavidin antibody. This was followed by another round of SAPE prior to scanning using a GeneChip Scanner 3000 (Affymetrix).
Scanning was performed using a GeneChip Scanner 3000 (Affymetrix)
After hybridization and quality control, probesets were filtered based on the following criteria: First, probesets were eliminated that were not classified as present in all arrays of any one combination of sex and treatment using PMA calls. Second, four separate normalizations of the data (MAS 5, RMA, GCRMA, and PLIER) were performed, and only those probesets that were above the 95th percentile of the background for all four normalizations were retained. The distribution of background intensity values in each case was determined using the negative control probes on the microarray. Ultimately, 6,165 probesets passed the filter criteria and were used in subsequent analyses. Arrays were then clustered based on expression levels of all expressed genes in our filtered set to test for the presence of outlier arrays. In this case, one sample (a male from the 25% carbohydrate treatment) did not cluster with the other arrays and was removed out of concern that this sample harbored contamination from other tissues.