Lung tissues were homogenized in the presence of TRI reagent (MRC Molecular Research Center, Cincinnati, OH). Total RNA was isolated using the 1-bromo-3-chloropropane (BCP) phase separation reagent (MRC Molecular Research Center). RNA was precipitated by 2-propanol and washed with 75% ethanol.
Label
biotin
Label protocol
Biotinylated cRNA was synthesized by in vitro transcription using the GeneChip IVT Labeling kit (Affymetrix).
Hybridization protocol
Biotin-labeled cRNA was purified (GeneChip Sample Cleanup Module, Affymetrix), and 20 mg of the labeled cRNA was fragmented. The cRNA and fragmented cRNA quality was assessed by the Agilent 2100 Bioanalyzer (Agilent; Foster City, CA) and the RNA 6000 Nano LabChip kit (Agilent). The labeled fragmented cRNA was hybridized to Affymetrix GeneChip Rat 230 2.0 arrays for 16 h at 45oC following the Affymetrix protocol specific to this array type. Washing and staining were performed on the Affymetrix fluidics (450) station according to the antibody amplification protocol (Fluidics script: EukGE-WS2v5).
Scan protocol
The Gene Chips were scanned using the GeneChip Scanner 3000 (Affymetrix).
Data processing
The analysis was performed with GeneSpring software package (Agilent Technologies, Santa Clara, CA). All samples were subject to per-chip normalization. Data were filtered twice, a fold change filter (2 fold up or down), and the Statistical Analysis of Microarray software (http://www-stat.stanford.edu/~tibs/SAM/), to find all genes with a < 10% false positive q-score.
