*4.5. RNA Isolation and Quantitative Real-Time PCR*

Total RNA was extracted from placenta tissue and JEG-3 cells using the trizol reagent (Invitrogen, United Kingdom). cDNA was synthesized using the PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Japan), following the manufacturer's instructions. Real-time PCRs were performed in 20 µL mixtures using the SYBR Premix Ex TaqTM II Kit (Takara, Japan). Gene expression was assayed in duplicate and normalized against β-actin. Relative expression values were calculated by the ∆∆CT method of relative quantification using Applied Biosystems 7500 Real-Time PCR System.
