Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay

Total RNA of the lung tissue was obtained using TRIzol reagent. The reverse transcription reactions for mRNA were performed using PrimeScript™ RT Master mix (Promega Corporation, Madison, WI, USA). The RT-qPCR analysis was performed using Taq polymerase (Takara Biotechnology Co., Ltd., Dalian, China) consisting of a final volume of 20 µl, containing 2 µl cDNA, 10 µl SYBR-Green Mix, 4 µl primer mix and 4 µl ddH2O (Takara Biotechnology Co., Ltd.). Primers specific for the TNF-α, IL-6, IL-1β, glyco-protein-B (gB) gene and β-actin are listed in Table I. β-actin was selected as an internal control to normalize target gene expression. Thermocycling conditions were as follows: 95°C for 5 min followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec, then a melting curve analysis from 60°C to 95°C every 0.2°C for 1.5 min was obtained. The transcript amount was normalized to U6 and β-actin and quantified using the 2^−ΔΔCq method (17).

Oligonucleotide primers used for reverse transcription-quantitative polymerase chain reaction analysis.

TNF-α, tumor necrosis factor-α; IL, interleukin; gB, glycoprotein-B.