Quantitative real-time reverse transcription PCR

After euthanasia, lungs were lavaged three times with 1mL of HBSS and centrifuged. The bronchus alveolar lavage fluid (BALF) supernatants were discarded and the cell pellets used for further assays. Total RNA was extracted from BALF cell pellet lymphocytes by applying Trizol (TransGen Biotech, Beijing, China) and subsequently treating with DNA-free kit to filter DNA contamination. First strand cDNA was transcribed using TransGen Reverse Transcription kit (TransGen Biotech, Beijing, China) according to the manufacturer’s protocols. Relative quantification of IL-2, IL-6, IL-10, IFN-γ was performed using SYBR Green PCR Master Mix kit (Takara, Dalian, China), The 2^−ΔΔCt method was used to calculate relative gene expression levels. Gene expression was assayed quantitatively and standardized to the level of a housekeeping gene (GAPDH) to obtain a RNA ratio in order to establish relevant change in RNA expression³¹.