mRNA expression analysis by quantitative real-time PCR

Cardiac fibroblasts (5 × 10⁵ cells/well) were plated in six-well plates. Cells were starved of serum for overnight before stimulation. Cells were pretreated without or with specific inhibitors/antagonists for 1 h before treatment with CV1808 for 1 h and further stimulated with 200 nM Ang II for 6 h. Cells were washed with phosphate buffer saline (PBS) and then performed the isolation of RNA using GeneJET RNA purification kits (Thermo scientific). Quantitative mRNA levels were performed using the KAPA SYBR FAST One-step RT-qPCR kits (KAPA biosystems) and the Mx 3005p Real Time PCR system (Stratagene) as described previously [12]. All sequences of primers are shown in Supplemental Table 1. Relative mRNA levels were calculated by the comparative cycle threshold (C_T) method and normalized to the endogenous reference, GAPDH relative to a calibrator.