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