Quantifying Gene Expression in Lung Samples

Frozen left lung samples (∼100 mg) were homogenized in TRIzol (Thermo Fisher Scientific, Waltham, MA, United States). The RNA was resuspended in nuclease-free water (20 μl) to later determination of its concentration and purity by spectrophotometry, being the 260/280 ratio of all samples in the range of 1.9–2.0. The RNA was stored at −80°C until later use. Synthesis to cDNA was performed using the cDNA Synthesis Kit (ABM, Richmond, Canada). The gene expression of TRPC1, TRPC4, TRPC6, ORAI1, ORAI2 and 18S was determined by real-time PCR (qPCR) in a StepOne Plus equipment (Applied Biosystems, Foster City, CA, United States), using a KicqStart kit (Sigma-Aldrich, United States) with the primers sequence used by Castillo-Galán et al. (2020) (link). Relative gene expression was calculated with the 2^–ΔΔCt method using 18S gene.