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).