Placental Gene Expression in Preeclampsia

Placentas obtained from pregnant women with PE and NT were submitted to analysis of the protein-encoding genes LC3-II, beclin-1, and mTOR. Total RNA was extracted from the placentas with the Total RNA Purification Kit (NorgenBiotek Corp., Canada) according to the manufacturer's protocol. After extraction, 1 µg of total RNA was incubated with DNase I Amp Grade (Invitrogen, ThermoFisher Scientific, USA), and measured by Qubit^® Fluorometric Quantitation (ThermoFisher Scientific). Subsequently, complementary DNA (cDNA) was synthesized using the ImProm-II^TM Reverse Transcription System (Promega, USA) according to the manufacturer's protocol. Quantitative real-time PCR (qPCR) was performed using RT GoTaq^® qPCR Master Mix (Promega) according to Matias et al. (20 (link)). A 7500 Fast Real-Time PCR System (Applied Biosystems - ThermoFisher Scientific, USA) was used for the analysis. For normalization, GAPDH was used. The primers used in the experiments are listed in Table 1. The differential expression calculation of selected genes was carried out by the data processing method compared with a standard curve (21 (link)). To analyze relative gene expression, RNA expression levels in all samples were standardized on a single RNA sample, which was set to a value of 100.