Quantitative Analysis of miRNA and mRNA Expression in BAL Cells

The gene expression for each miRNA and mRNA in BAL cells was investigated by qRT-PCR using specific primers and probes (see Table S1 and Table S2, in Supplementary Material available online at http://dx.doi.org/10.1155/2015/121378). qRT-PCR for each individual miRNA was performed in a 20 μL reaction mixture that included 1.3 μL of diluted RT product, 1 μL of 20X TaqMan Individual microRNA assay, 10 μL of 2X TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems), and 7.7 μL of nuclease-free water. qRT-PCR for each individual mRNA expression was performed as described previously [21 (link)]. All reactions were performed on RotorGene3000 system (Qiagen Inc., Valencia, CA, USA); the reaction steps were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. The relative miRNA and mRNA expression levels were calculated by a second-derivative method (RotorGene Software 6.1.81, Corbett, Sydney, Australia); cDNA from human universal reference RNA (Stratagene, La Jolla, CA, USA) was used as a calibrator. A reference gene for miRNA analysis was endogenous control Mammalian U6 and for mRNA a reference gene PSMB2 [21 (link)]. Changes in expression levels are presented as mean relative expression with 95% confidence interval (CI).