Quantitative Real-Time PCR Analysis of Gene Expression in Lung Tissue

Total RNA from day 12 lung tissue (40 mg) previously immersed in liquid nitrogen and pulverized with a mortar pestle on ice was extracted using the Aurum Total RNA Mini-Kit (Bio-Rad Laboratories, Hercules, CA) and quantified (Nanodrop Spectrophotometer, ThermoScientific, Waltham, MA). RNA (2 μg) exhibiting A_260/280 values of 1.8–2.0 was converted to cDNA using the Reliance Select cDNA Synthesis Kit (Bio-Rad) with random primers as previously described by Arkatkar and coworkers [15 (link)]. The resulting cDNAs were further amplified using the SsoAdvanced PreAmp Supermix (Bio-Rad). Quantitative real-time PCR was carried out using cDNA and gene-specific primer pairs (GAPDH qMmuCED002749; HSP90ab1 qMmuCED000500; CCL3 qMmuCED0044190; HMGB1 qMmuCED0041193; SOCS1 qMmuCED0024846; TBX21 qMmuCID0022343; IL-4 qMmuCED0044969, and SMS qMmuCED0040754 (Bio-Rad)) dissolved in SsoAdvanced Universal SYBR Green Supermix (Bio-Rad), and a CFX 96 instrument was used (Bio-Rad). All mRNA expression levels were normalized to housekeeping genes GAPDH and HSP90 and reported as expression relative to that of WT naïve mouse expression using the comparative cycle threshold method [16 (link)]. Quantification of gene expression was achieved using the CFX Maestro software (Bio-Rad), and results were reported as fold-change differences as previously described [14 (link), 15 (link)].