4.5. Quantitative Real-Time PCR

Total RNA was isolated from cultured NHDFs using RNAiso Plus (Takara Bio Inc., Shiga, Japan). One microgram of total RNA was converted to cDNA using the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative real-time PCR was performed on a 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using TB green™ Premix Ex Taq™ (Cat# RR420A, Takara Bio) according to the manufacturer’s instructions. The primer sequences were listed in Table S1. The PCR conditions were 50 °C for 2 min, following by 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. The data were analyzed by the 2^{(−∆∆Ct)} method and represented as fold changes of gene expression relative to 36B4.