2.4. RNA isolation from SAECs, BSMCs and sputum cells, reverse‐transcription, and qPCR

RNA was purified from SAECs, BSMCs, or sputum cells by utilizing the phenol‐chloroform technique from the QIAzol Lysis reagent (Qiagen). 500 ng of total RNA quantified by Nanodrop ND‐1000 spectrophotometer (Thermo Fisher Scientific) was reverse‐transcribed using the High‐Capacity cDNA Reverse Transcription Kit (Applied Biosystems), followed by semi‐quantitative real‐time PCR (qPCR) following manufacturer's guidelines on a 7500 Real‐Time PCR System with TaqManTM gene expression probes for POSTN, RHOA, CDC42 and GAPDH and TaqManTM Gene Expression MasterMix (Applied Biosystems). Alternatively, 100 ng of RNA were retrotranscribed using a miRCURY LNA™ RT Kit (Qiagen) followed by qPCR in a Light Cycler 96 thermocycler (Roche) using miRCURY LNA™ SYBR Green PCR Kit and hsa‐miR‐185‐5p, miR‐191‐5p and U6, and UniSp6 probes (Qiagen). MiR‐191‐5p was used as housekeeping miRNA for BSMCs and U6 for SAECs/sputum. Relative gene expression was calculated using the Cycle Threshold (Ct) and the 2‐ΔΔCt method (Livak & Schmittgen, ²⁰⁰¹), where: