Quantification of miR-141 and its transcripts

Total RNA was isolated from tissue samples and cell lines using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. The levels of mature miR-141 (miR-141) and precursor miR-141 (pre-miR-141) were evaluated using the miDETECT A Track miRNA qRT-PCR Kit (RiBoBio). The U6 small nuclear RNA (RNU6B) (RiBoBio) was used for normalization. The expression of primary miR-141 transcript (pri-miR-141), c-Myc and BRD7 was measured by qRT-PCR according to the instructions of the SYBR Premix Ex Taq (TaKaRa, Dalian, China). The GAPDH mRNA level was used for normalization. The relative expression ratio was calculated using the 2^−ΔΔCT method. The primers for miR-141, pre-miR-141, U6, pri-miR-141, c-Myc, BRD7, and GAPDH were described previously [23 (link)]. PCRs of each sample were conducted at least in triplicate.