RNA Extraction and RT-PCR Analysis

Total RNA was isolated from placental tissues or BeWo cells using EZNA. Total RNA Kit (OMEGA, Georgia, USA) according to the manufacturer's instructions. Reverse transcription to synthesize cDNA was accomplished using PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Shiga, Japan). PCR amplification of the cDNA was performed using specific mouse primers shown in electronic supplementary material, figure S1. PCR was performed in a BIO-RAD S1000 Thermal cycler (BIO-RAD). The cDNAs were amplified for 40 cycles. One round of amplification was performed at 95°C for 5 s, at 56°C for 30 s and at 72°C for 30 s (TaKaRa, Japan). The PCR products (20 µl) were resolved on 2% agarose gels (Biowest, Spain) in a 1× TAE buffer (0.04 M Trisacetate and 0.001 M EDTA) and with GeneGreen Nucleic Acid Dye (TIANGEN, China). The reaction products were visualized using a transilluminator (SYNGENE, UK) and a computer-assisted gel documentation system (SYNGENE). The sets of primers used for RT-PCR are provided in the electronic supplementary material, figure S1. The ratio between the intensity of the fluorescently stained bands corresponding to the genes and β-actin was calculated to quantify the level of the transcripts for those genes mRNAs [15 (link),22 (link)]. The RT-PCR result was representative of three independent experiments.