2.5 Total RNA extraction and gene expression assay
Total RNA extraction from the placental, adrenal, and NCI-H295R cells was performed using TRIzol reagent (Invitrogen Co.; 15596018) and following the manufacturer’s protocol. The concentration and purity of the isolated RNA were determined by a spectrophotometer (NanoDrop 2000C, Thermo), and the RNA concentration was adjusted to 1 μg/μl. Complementary DNA was synthesized from 1 μg of total RNA according to the kit protocol and was stored at −20°C for testing. RNA preparations were reverse transcribed to generate cDNA using the Reverse transcription reagent kit (Takara Biotechnology Co., Dalian, China) and miScript Reverse transcription kit (RiboBio, Guangzhou, China), respectively. Quantitative measurements of mRNA and miR expression were done using a SYBRGreen PCR kit (Applied Biosystems, ABI, 4472908) and miDETECT A Track™ miRNA quantitative real-time polymerase chain reaction (qRT-PCR) kit (RiboBio, Guangzhou, China) according to the manufacturer’s guidelines and a StepOnePlus Real-time PCR system (Applied Biosystems, Foster City, CA, USA), using specific primers. U6 snRNA was used as an internal control for the miRNAs, GAPDH was used as an internal control for the adrenal samples, and calculated by the cycle threshold 2△△Ct method. The sequences of primers used in this experiment are shown in Table S1. All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).