Quantification of Lung mRNA Transcripts

Total RNA was isolated from murine lung tissue or cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. Briefly, the RNA precipitate was washed twice by gentle vortexing with 70% ethanol, collected by centrifugation at 12000 rpm, dried under a vacuum for 5–10 min, dissolved in 200 μl RNase-free water (Promega, Madison, WI, USA), and incubated for 10–15 min at 55–60 °C. The RNA was quantified and assessed for purity by spectrophotometry at a wavelength of 260 nm. The integrity was assessed by 2% agarose gel electrophoresis and the RNA was visualized by ethidium bromide staining. The GABA_AR subtype and Muc5ac mRNA transcripts were measured by RT-PCR as previously described30 (link)52 (link). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous control gene. The PCR primers for mice and humans were designed according to the published cDNA sequences and synthesized by MDBio Inc (Taipei, Taiwan). The primers sequences are listed in Table 1. Mouse or human GAPDH mRNA was used as the internal control in all experiments. The mRNA expression levels of Muc5ac and GABA_AR subunits were measured by band intensities using the Gel Analysis method in ImageJ 1.49 and were normalized to the level of GAPDH.