Placental Gene Expression Analysis

Total RNA was extracted from the placentas using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA (1 μg) was used for reverse transcription using high-capacity cDNA of the reverse transcription kit RT (TaKaRa Biotechnology CO., Ltd) according to the manufacturer’s instructions. The PCNA, Cyclin D3, Slc38a1/SNAT1, Slc38a2/SNAT2, Slc38a4/SNAT4, TAUT, Slc2a1/GLUT1, Slc2a3/GLUT3 and GAPDH mRNA primer sequences were designed according to earlier publications15 (link)50 (link)51 (link). RT-qPCR was performed using a Light Cycler Fast Start DNA Master SYBR Green I Kit and an ABI Prism 7300 Sequence Detection System (Applied Biosystems, Foster City, California, USA), and relative gene expression was determined using the 2-ΔΔct method with normalization to GAPDH expression. The results were averaged from four sets of independent experiments.