Quantitative real-time RT-PCR (qPCR)

Total RNA was extracted from lungs of mice at 21 days after receiving BLM (n = 5 per group). After RNA extraction with an RNeasy Mini Kit (Qiagen Ltd., Manchester, UK), cDNA was synthesised using an ExScript RT Kit (Takara, Shiga, Japan) and amplification was performed in a Sequence Detection System 7000 (Thermo Fisher Scientific Inc., MA, USA) according to the manufacturer’s instructions. The primer sequences for matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), tissue inhibitor of metalloproteinase-2 (TIMP-2), collagen type 1 alpha 1 (COL1A1), hepatocyte growth factor (HGF), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), and glyceraldehyde 6-phosphate dehydrogenase (GAPDH) as a housekeeping gene are summarised in Table ​Table1.1. The amount of each target gene expression was normalised to the housekeeping gene expression. The following PCR conditions were used: 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, 56 °C for 30 s, and 95 °C for 10 s, and a final extension at 95 °C for 50 s. The relative mRNA expression level of each target gene was calculated by the comparative CT method²¹. The experiments were carried out five times independently for each sample, and in duplicate.

Primers used in the qPCR analyses.