Quantitative Real-Time PCR for Gene Expression Analysis

qRT-PCR was performed as previously described [27 (link)]. Total RNA was isolated using ISOSPIN Cell & Tissue RNA (Nippon Gene, Tokyo, Japan). Then, 100 ng of total RNA was transcribed to cDNA using the TaqMan Universal Master Mix. Mouse beta-actin (4352341E, Applied Biosystems, Foster City, CA, USA) was used as the endogenous control. qPCR was performed using a QuantStudio 12K Flex instrument (Applied Biosystems). The primers and probes were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The assay identification of each primer and probe are as follows: bone morphogenetic protein receptor type-2 (BMPR2; Mm03023976_m1); transforming growth factor-b1 (Tgf-b1; Mm 01178820_m1); plasminogen activator inhibitor-1 (PAI-1; Mm00435860_m1); interleukin-6 (IL-6; Mm00446190_m1); and tumor necrosis factor (TNF; Mm00443258_m1).