RNA extraction and quantitative reverse transcription PCR analysis

Total RNA was extracted from frozen samples or harvested cells, using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). To analyse miR‐140‐3p, miR‐193a‐3p and miR‐22‐3p expression, reverse transcription was performed using specific stem‐loop primers, using the ImProm‐IITM Reverse Transcription System (Promega, Madison, WI, USA). The ImProm‐IITM Reverse Transcription System was also used to quantify the mRNA level of CYP11B2. Fluorescence‐based quantitative reverse transcription PCR (qRT‐PCR) was performed using SYBR GREEN qPCR Super Mix (Invitrogen) on an Applied Biosystems 7500 system. U6 was used as an internal control for normalization of miRNA expression. 18S rRNA was used as an internal control for normalization of CYP11B2 expression. The primer sequences used for qRT‐PCR are shown in Table 1. Gene expression was measured in triplicate, quantified using the 2^−ΔΔCT method and normalized using the relevant control.

Primers for quantitative real‐time RT‐PCR

F, Forward primer; R, reverse primer.