Total RNA was extracted (TRIzol; Invitrogen, Carlsbad, CA) and purified (RNeasy Mini Kit; Qiagen, Valencia, CA). RNA quality was checked by gel electrophoresis. The first strand of cDNA was synthesized with 10 μg of total RNA, 1 μL of T7-(dT)24 primer (100 picomoles/μL; Invitrogen) and a reverse transcriptase (Superscript II RNase H Reverse Transcriptase kit; Invitrogen), as per the manufacturer’s instructions. Second-strand cDNA synthesis was performed by adding the single-strand cDNA solution DEPC-H2O (91 μL), 5× second-strand buffer (30 μL; Invitrogen), 10 mM dNTP mix (3 μL; Invitrogen), 10 U/μL Escherichia coli DNA ligase (1 μL; Invitrogen), 10 U/μL E. coli DNA polymerase I (4 μL; Invitrogen), and 2 U/μL E. Coli RNase H (1 μL; Invitrogen) and incubated at 16°C for 2 hours. T4 DNA polymerase (2 μL; Invitrogen) was then added and the solution incubated in a 16°C water bath for 5 minutes. The reaction was then stopped by adding 10 μL of 0.5 M EDTA (Ambion, Austin, TX). The double-stranded cDNA was purified with a pre-spin phase-lock gel (PLG) tube (VWR Scientific; Chicago, IL) run for 30 seconds at 12,000g. Phenol-chloroform-isoamyl alcohol (25:24:1; 162 μL; Invitrogen) was added to the cDNA, vortexed briefly, and rerun through the PLG tubes for 2 minutes at 12,000g. The aqueous phase was transferred to a fresh 1.5 mL tube, and diluted 2:1 with 7.5 M NH4OAc (Sigma-Aldrich, St. Louis, MO) and this mixture diluted 1:2.5 with absolute ethanol. This mixture was vortexed thoroughly and centrifuged at room temperature for 20 minutes at 12,000g. The pellet was washed twice with 0.5 mL of 80% ethanol. After it was air dried, the pellet was resuspended in RNase-free water and quantified by gel electrophoresis.
Biotin-labeled complementary RNA (cRNA) was synthesized with an RNA transcript labeling kit (BioArray High Yield RNA kit; Enzo Life Sciences, Farmingdale, NY) according to the manufacturer’s instructions. Product was purified and quantified as indicated above. Fragmentation of the cRNA was performed with a kit (GeneChip Sample Cleanup Module Kit; Affymetrix, Inc., Santa Clara, CA).
Fragmented cRNA was hybridized to the Rat U34 GeneChip (Affymetrix, Inc.), according to the manufacturer’s instructions. After hybridization, the chips were run through a fluidics station and scanned through a spectrophotometer (both from Affymetrix).
We then subjected the microarray data to a robust multiarray analysis protocol
10 and false-discovery-rate analysis,
11 as previously described.
12 13 This allowed probe level background adjustments and quantile normalization and assigned log-transformed perfect-match values to each gene. Genes were then categorized as having significant changes in transcript abundance with a false-discovery rate set at less than or equal to 50% (i.e., the probability that an identified gene is actually differentially expressed is 50%).