2.7. Real Time PCR (RT-PCR)

Placental tissues, trophoblast cells, and Trizol (Vicmed, Busan, Korea) were used to isolate total RNA. RNA concentration was detected by Nanodrop 2000 (Thermo, Scientific). The reverse transcription was conducted using SYBR Green qPCR SuperMix and 500 ng of the total RNA was reverse transcribed to cDNA. The quantitative RT-PCR was carried out using SYBR Green Master Mix on a 7500 fast real-time PCR System (Applied Biosystem, Foster, CA, USA) according to the manufacturer’s instructions. The mRNA levels were analyzed using the comparative cycle threshold method (2^−ΔΔCT), and the relative levels were normalized to the level of GAPDH. The primers were designed by Sangon Biotech (Shanghai, China), and primer sequences were listed in Supplementary Table S1.