Determination of cytokine expression by fluorescent quantitative PCR.

Lung tissue was ground in liquid nitrogen, RNA was extracted based on the kit manufacturer's instructions, and cDNA was prepared using a reverse transcription kit (TaKaRa, Tokyo, Japan). The PCR system included 10 μl SYBR green (TaKaRa, Tokyo, Japan), 7.4 μl water, 0.8 μl primer, and 1 μl template, and the expression levels of IL-6, IL-10, IL-18, and TNF-α were determined. All primers are listed in Table 1.

Primers used for RT-qPCR