Total RNA was prepared using the RNeasy Kit (Qiagen) as per the manufacturer’s instructions. cDNA was prepared and assayed using Superscript III (Sigma) according to the manufacturer’s protocol. Differential gene expression was determined using SYBR green detection (Roche). Real-time PCR reactions were done in triplicate for each sample. GAPDH was used as a housekeeping gene to normalize gene expression levels using the LightCycler 480 software (Roche). Normalized mRNA levels are shown as relative to the control samples (day 0 fresh isolated cells or adult lung).
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