One microgram of total RNA from each sample was used to generate complementary DNA (cDNA) by using M-MLV reverse transcriptase as per the manufacturer’s specifications (Promega Corporation, USA). Real-time PCR was cycled in 95°C/15 s, 60°C/30 s, and 72°C/30 s for 40 cycles, after an initial denaturation step at 95°C for 10 min using SYBR Green PCR Master Mix purchased from Applied Biosystems (USA). Amplification was performed by using 7500 Fast Real-Time PCR Systems (Applied Biosystems, USA), and the products were routinely checked by using dissociation curve software. Transcript quantities were compared by using the relative quantitative method, where the amount of detected mRNA was normalized to the amount of endogenous control (GAPDH). The relative value to the control sample is given by 2−ΔΔCT.

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