Fast sybr green master mix

Total RNA was extracted using TRI reagent (MilliporeSigma) and Direct-zol™ RNA miniprep (Zymo Research Corp.). RNA (1 µg) was then reverse transcribed into cDNA using an RT kit (sensiFAST; Bioline) according to manufacturer's protocol. Fast SYBR Green Master Mix (MilliporeSigma) was used to perform qPCR with a final concentration of 0.25 nM gene-specific primers. The sequences of the primers were as follows: E-cadherin forward: 5′-CCGAGAGCTACACGTTC-3′, reverse: 5′-TCTTCAAAATTCACTCTGCC-3′; fibronectin-1 forward: 5′-CCATAGCTGAGAAGTGTTTTG-3′, reverse 5′-CAAGTACAATCTACCATCATCC-3′; MMP2 forward: 5′-GTGATCTTGACCAGAATACC-3′, reverse: 5′-GCCAATGATCCTGTATGTG-3′; thrombospondin 1 (THBS1) forward: 5′-GTGACTGAAGAGAACAAAGAC-3′, reverse 5′-CAGCTATCAACAGTCCATTC-3′; and zinc-finger E-box-binding homeobox 1 (ZEB-1) forward: 5′-AAAGATGATGAATGCGAGTC-3′, reverse: 5′-TCCATTTTCATCATGACCAC-3′. PCR signals were compared among groups after normalization, with GAPDH forward: 5′-TCGGAGTCAACGGATTTG-3′, reverse: 5′-CAACAATATCCACTTTACCAGAG-3′) used as the internal reference, calculated using the 2^−ΔΔCq method (18 (link),34 (link),35 (link)). The thermocycling conditions were as follows: 95 °C for 10 min. Next, they were denatured at 95°C for 15 sec and annealing the primer with DNA template at 60°C for 1 min. These cycles were repeated for 40 times.