Total RNA was isolated from approximately 50-100 mg of tissue from each animal using RNA STAT-60 according to the manufacturer's instructions (Tel Test Inc) [RNA Stat-60 is the same as Trizol reagent].
Label
biotinylated UTP & CTP
Label protocol
First and second strand cDNA were synthesized from 15 ug of total RNA using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen) and oligo-dT24-T7 (5’-GGC CAG TGA ATT GTA ATA CGA CTC ACT ATA GGG AGG CGG-3’) primer (PrOligo) according to the manufacturer’s instructions. cRNA was synthesized and labeled with biotinylated UTP and CTP by in vitro transcription using the T7 promoter-coupled double stranded cDNA as template and the Bioarray™ HighYield™ RNA Transcript Labeling Kit (ENZO Diagnostics Inc.). Briefly, double stranded cDNA synthesized from the previous steps were washed twice with 70% ethanol and resuspended in 22 ul RNase-free H2O. The cDNA was incubated with 4 ul each of 10X Reaction Buffer, Biotin-labeled Ribonucleotides, DTT, RNase Inhibitor Mix and 2 ul 20X T7 RNA Polymerase for 5 hr at 37oC. The labeled cRNA was separated from unincorporated ribonucleotides by passing through a CHROMA SPIN-100 column (Clontech) and ethanol precipitated at –20oC for 1 hr to overnight.
Hybridization protocol
The cRNA pellet was resuspended in 10 ul RNase-free H2O and 10.0 ug was fragmented by ion-mediated hydrolysis at 95oC for 35 min in 200 mM Tris-acetate (pH 8.1), 500 mM potassium acetate, 150 mM magnesium acetate. The fragmented cRNA was hybridized for 16hr at 45oC to GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix), which cover over 47,000 human transcripts and variants representing approximately 39,000 of the best characterized human genes (Affymetrix). Arrays were washed at 25oC with 6 X SSPE (0.9M NaCl, 60 mMNaH2PO4, 6 mM EDTA + 0.01% Tween 20) followed by a stringent wash at 50oC with 100 mM MES, 0.1M [Na+], 0.01% Tween 20. The arrays were then stained with phycoerythrein-conjugated streptavidin (Molecular Probes) and the fluorescence intensities were determined using the GCS 3000 high-resolution confocal laser scanner (Affymetrix).
Scan protocol
The arrays were then stained with phycoerythrein-conjugated streptavidin (Molecular Probes) and the fluorescence intensities were determined using the GCS 3000 high-resolution confocal laser scanner (Affymetrix). The scanned images were analyzed using programs resident in GeneChip Operating System (GCOS; Affymetrix). Sample loading and variations in staining were standardized by scaling the average of the fluorescent intensities of all genes on an array to a constant target intensity of 250 for all arrays used.
Description
The expression data were analyzed by MAS5 statistical algorithms resident in GCOS; in short, the signal intensity for each gene was calculated as the average intensity difference, represented by [Σ(PM - MM)/(number of probe pairs)], where PM and MM denote perfect-match and mismatch probes (Lockhart et al, 1996, Nat. Biotechnol. 14:1675-1680; Liu et al, 2002, Bioinformatics 18:1593-9; Hubbell et al, 2002, Bioinformatics 18(12):1585-92). GCOS-generated detection and change calls were used to identify robust expression changes.
