Moloney murine leukemia virus reverse transcriptase

Total RNA was extracted with Trizol Reagent (Sangon Biotech, Shanghai, China) according to the protocol of the manufacturer. RNA quality was examined by electrophoresis on a 2.0% agarose gel, and its amount was analyzed by a spectrophotometer (NanoDrop 2000, Thermo Scientific, Waltham, MA, USA). For RT-qPCR analysis, cDNA was synthesized with the Moloney Murine Leukemia Virus Reverse Transcriptase (Sangon Biotech, Shanghai, China) according to the protocol of the manufacturer. The reaction volume was set to 20 μL in accordance with the operation manual of the 2xSG Fast qPCR Master Mix (Sangon Biotech, Shanghai, China). DNA was amplified with a real-time PCR system (QuantStudio 1, Applied Biosystems by Thermo Fisher Scientific, Bend, OR, USA) to analyze the expression of target genes under the following PCR procedure: denaturation of 3 min at 95 °C and 30 cycles of 95 °C for 10 s and 55 °C for 30 s and 72 °C for 60 s. The primers used for amplification of the reference gene RLPL32 and target genes are presented in Table 1.

miR-96 and U6 snRNA-specific cDNA was formed using the Gene Amp System 9700 (Applied Biosystems, Foster City, CA, USA). U6 snRNA was used as an internal control. Reverse transcriptase primers for miR-96 and U6 were 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACAGCAAAA-3′ and 5′-CGCTTCACGAATTTGCGTGTCAT-3′. Reverse transcription conditions contained 800 ng total RNA, 0.3 μl stem-loop RT primer (1 μM), 2 μl 10× RT buffer, 2 μl dNTP (2.5 mM each), 0.2 μl Moloney murine leukemia virus reverse transcriptase (Sangon, Beijing, China), and 0.3 μl RNase inhibitor (40 U/μl). The 20-μl reaction volumes were incubated at 16°C for 30 min, 42°C for 40 min, 85°C for 5 min, and then held at 4°C or stored at −20°C.