Quantitative reverse transcription PCR

To isolate total RNA from lungs, left main bronchial rings and cells were homogenized directly in 700 μl of lysis/binding buffer provided by the miRNeasy Mini Kit (QIAGEN). Total RNA was transcribed into complementary DNA (cDNA) using a TaqMan microRNA reverse transcription kit (Life Technologies), and qPCR was performed using cDNA with TaqMan Universal Master Mix II (Life Technologies), according to the manufacturer’s instructions. The assay numbers for endogenous control miR [U6 snRNA (small nuclear RNA)] and target miR (hsa–miR-133a) were 1973 and 2246, respectively. cDNA was generated using a T100 thermal cycler (Bio-Rad), and reverse transcription PCR was performed using an iCycler iQ system (Bio-Rad). Relative expression of the transcripts was determined according to the ΔΔC_t method using U6 snRNA as reference for normalization.