Total RNA was isolated from each biopsy sample using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The RNA was purified using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol.
Label
biotin
Label protocol
RNA amplification and microarray analysis for both experiments was performed at the University of Vermont microarray core facility using previously described protocols (Affymetrix, 2005-2006). Briefly, 2 ug of total RNA from each tissue sample were reverse transcribed to the single stranded cDNA using a T7-oligo(dT) primer. T4 DNA polymerase was used to synthesize double-stranded cDNA, which served as a template for in vitro transcription using T7 RNA polymerase to produce biotinylated cRNA.
Hybridization protocol
The biotinylated cRNAs were fragmented into 50- to 200-base fragments and then hybridized to GeneChip Bovine Genome Arrays for 16h at 45°C in a rotating Affymetrix GeneChip Hybridization Oven 320. After hybridization, arrays were washed and stained with streptavidin-phycoerythrin on an automated Affymetrix GeneChip Fluidic Station F450.
Scan protocol
The arrays were scanned with an Affymetrix GeneChip Scanner 2700 and the images quantified using Affymetrix GeneChip Operating Software.
Data processing
The signal intensity for each probe on each chip was calculated from scanned images using GeneChip Operating Software (Affymetrix), and signal intensities were analyzed using BioConductor (http://www.bioconductor.org). Probe intensities were background corrected, normalized, and summarized using the Robust Multichip Average method described by Speed and coworkers (Bolstad et al., 2003; Irizarry et al., 2003). An alternative normalization method based on housekeeping genes did not significantly change the results. The false discovery rate (FDR) was calculated by performing distinct permutations of the sample labels (Benjamini et al., 2001; Storey and Tibshirani, 2003; Storey et al., 2005). In experiment 1, probe sets were considered differentially expressed when the signed fold change (4X vs. 2X) was ≥ 2, the P-value was < 0.05, and the FDR was < 0.20. In experiment 2, probe sets were considered differentially expressed when the signed fold change was ≥ 1.2 and the P-value was < 0.10. The less stringent signed fold change and P-value cutoffs for experiment 2 were used to allow for identification of genes that were differentially expressed in experiment 1 and may have been differentially expressed in experiment 2, but to a lesser degree. To protect against false positives, the results of experiment 1 were used as a filter and only those genes differentially expressed in that experiment were investigated for differential expression in experiment 2.
