Quantitative real-time PCR
Total RNA (500 ng) was extracted using RNeasy mini kit (Qiagen)
according to the manufacturer’s instructions. Total RNA (500 ng) was
reversed transcribed with Primescript RT (Takara) and the cDNA served as
template for amplification and quantitative real-time PCR, which was
performed using SYBR Green Master Mix (TaKaRa) on a LightCycler480
Roche. Relative quantification was measured using the Comparative Ct
(Threshold Cycle) and normalizing the Ct values to GAPDH. All samples
were analyzed in duplicate and the mean of the 2 runs were reported.