Real-Time PCR

Cells were cultured and treated as previously described in a 12-well plate. Total RNA was extracted from cells by RNAiso, and cDNA was obtained using a Reverse Transcription System (Takara). Real-time PCR was conducted with TB Green Premix Ex Taq II (Takara), and primers are shown in Table 1. Melting curve analysis (60-95°C) was used for assessing amplification specificity. The PCR condition and cycling parameters were described recently [19]. Each reaction was performed in triplicate. Relative expression of each gene was calculated according to the 2^-ΔΔCt method with β-actin gene as an endogenous control [20].

Prime information for real-time PCR.