2.2 Tissue collection and microarray analysis
Total RNA was isolated from individual whole lungs (P21) using miRVana kits (Ambion) according to the manufacturer’s instructions and our previous report.1 RNA quality was controlled by screening for OD 260/280 ratios between 1.8 to 2 and for OD 260/230 ratio of 1.8 or greater. One microgram of total RNA was used, and three individual total RNA samples at the same time point were pooled as “single” sample in order to minimize both biologic variability and the number of microarrays necessary to generate informative data.1 miRNA expression analyses were performed using an Affymetrix Gene-Chip miRNA Array (Affymetrix, Genisphere, FT30AFYB), which included poly(A) tailing with RNA samples, flash tag ligation, and hybridization. The chips were scanned, and the signal intensity was computed (GeneChip Command Console 1.1 software, Affymetrix, Santa Clara, California) according to the manufacturer’s instructions and our previous report.1 Transcript expression analyses were performed using an Affymetrix Expression Array (Affymetrix, Mouse Genome 430 2.0 Array), which involved cDNA synthesis, cRNA synthesis, amplification, purification, and hybridization. The microarray chips were scanned and the imaged data were processed using GeneChip Command Console 1.1 software (Molecular Devices, USA Affymetrix, Santa Clara, California) according to the manufacturer’s instructions and our previous report.1