Crnde

Sequestering of total RNA was achieved by means of RNAzol reagent (Vigorous Biotechnology Beijing Co., Ltd.) as per the manufacturer's instructions. Then, Moloney murine leukemia virus reverse transcriptase (Promega Corporation) was used to reverse transcribe total isometric RNA (2 µg), with the transcription level normalized to the 18S rRNA level. Subsequently, RT-qPCR was performed using an ABI 7300 RT-PCR system with SYBR^® Green RT-PCR Master mix (Toyobo Life Science). The qPCR was conducted at 95˚C for 10 min followed by 40 cycles of 95˚C for 30 sec and 60˚C for 1 min. The primers for CRNDE, miR-3148 and DCUN1D1 were purchased from Guangzhou RiboBio Co., Ltd. The primer sequences that were used are as follows: miR-3148 forward (F), 5'-TGGAAAAAACTGGTGTGTGCTT-3'; miR-3148 reverse (R), 5'-GCTGTCAACGATACGCTACCTA-3'; DCUN1D1 F, 5'-AGGATCATTGGACAGGAAGAAGT-3'; DCUN1D1 R, 5'-TGCCAGGTCATCACAGAACTG-3'; GAPDH F, 5'-AGAAGGCTGGGGCTCATTTG-3'; and GAPDH R, 5'-AGGGGCCATCCACAGTCTTC-3' was used as an endogenous control for mRNA. The expression of miRNA was normalized to U6 F, 5'-CTCGCTTCGGCAGCACA-3'; and U6 R, 5'-AACGCTTCACGAATTTGCGT-3'. Finally, the relative content of specimens was examined using the 2^-ΔΔCq method (20 (link)). Each assay was averaged over three performances.