Super moloney murine leukemia virus m mlv reverse transcriptase

Total RNA was extracted from each sample using a total RNA extraction kit (BioTeke, Beijing, China) according to the manufacturer's protocol. The isolated total RNA was reverse-transcribed using a Super Moloney murine leukemia virus (M-MLV) reverse transcriptase (BioTeke). The reverse transcription mixture contained 1 µg RNA, 1.2 µl RT primer, 0.75 µl dNTP, 4 µl 5x Buffer, 0.25 µl RNasin, 0.2 µl M-MLV, and ddH₂O added up to 20 µl. The reaction conditions were as follows: 25°C for 10 min, 42°C for 50 min, then 80°C for 5 min. The reaction products were kept on ice. The miR-210 level in each sample was then detected by RT-qPCR (SYBR Green method). The RT-qPCR mixture contained 1 µl reverse transcription products, 0.5 µl forward primer, 0.5 µl reverse primer, 10 µl SYBR Green Mastermix (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China), and ddH₂O added up to 20 µl. The primers used were as follows: miR-210 forward, 5′-CATAGATAGCCACTGCCCACA-3′ and reverse, 5′-GTGCAGGGTCCGAGGTATTC-3′; U6 small nuclear (sn) RNA forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The cycling conditions were 95°C for 10 min, 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec, followed by a final incubation at 4°C for 5 min. The miR-210 level was normalized to U6 snRNA and the relative miR-210 level was calculated using the 2^−∆∆Cq method (22 (link)).

RPMI-1640 culture medium was obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA); fetal bovine serum (FBS) was obtained from HyClone Laboratories (Logan, UT, USA); the eukaryotic expression vector pEGFP-N1 and liposome 2000 were obtained from Invitrogen (Carlsbad, CA, USA); Super Moloney murine leukemia virus (M-MLV) reverse transcriptase was obtained from BioTeke (Beijing, China); RNA Simple Total RNA Kit and Total RNA Extraction Kit were obtained from Tiangen Biotech (Beijing, China); MTT reagent was obtained from Sigma-Aldrich (St. Louis, MO, USA); NP-40 lysis buffer, bicinchoninic acid (BCA) Protein Assay Kit and phenylmethylsulfonyl fluoride (PMSF) were obtained from Beyotime Biotechnology (Jiangsu, China); and electrochemiluminescence (ECL) luminescence reagent from 7sea Biotech (Shanghai, China).