Real-Time PCR Analysis of Placental Gene Expression

Real-time PCR was performed as previously described (3 (link)). Total RNA was extracted from placental tissues using RNeasy Plus Mini Kit (QIAGEN). One μg of total RNA was reverse-transcribed using qScript cDNA Synthesis Kit (Quantabio). The resulting cDNA was quantified by real-time PCR (CFX96 Real-Time System, Bio-Rad) using PerfeCTa FastMix II from Quantabio and mouse-specific TaqMan (Assays-on-Demand) probes targeting 18S (Mm03928990_g1), Vegfa (Mm01281449_m1), and Hyou1 (Mm00491279_m1), purchased from Applied Biosystems (Thermo Fisher Scientific). For each probe, a dilution series determined the efficiency of amplification of each primer set. Gene expression was normalized to 18S, expressed as the relative fold change using the ΔΔCt method, and compared with selected appropriate positive or negative controls.