Mmlv reverse transcriptase first strand cdna synthesis kit

Total RNA was extracted from cultured cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. miR-190b expression levels were detected using an ABI 7500 (Applied Biosystems Thermo Fisher Scientific, Inc.) using One Step TB Green^® PrimeScript™ RT-PCR Kit (Takara Bio, Inc.) after transcribing the extracted RNA into complementary DNA (cDNA) using MMLV Reverse Transcriptase First Strand cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 15 min, 85°C for 5s, and 4°C for 60 min. The sequences of the primers used were as follows: miR-190b forward, 5′-GGGTGATATGTTTGATAT-3′ and reverse, 5′-CAGTGCGTGTCGTGGAGT-3′; U6 small nuclear RNA forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; FOXP2 forward, 5′-AACAACAGCAGGCTCTCCAG-3′ and reverse, 5′-GGCACCTGCAGTGGTCTC-3′; and GAPDH forward, 5′-GGTGGTCTCCTCTGACTTCAACA-3′ and reverse, 5′-GTTGCTGTAGCCAAATTCGTTGT-3′. Expression levels of miR-190b or FOXP2 were calculated using the 2^−∆∆Cq method with U6 snRNA or GAPDH as endogenous controls (11 (link)). Experiments were performed in triplicate. The thermocycling conditions were as follows: Initial denaturation at 95°C for 30s, followed by 40 cycles of 95°C for 10s and 56°C for 30s.

Total genomic DNA was extracted from tissues by DNA digestion buffer incubation (50 mM Tris-HCl pH 8.0, 100 mM EDTA pH 8.0, 100 mM NaCl, 1% SDS, 0.5 mg/mL proteinase K) overnight at 65°C with gentle shaking, and then treated with 10 μg/mL RNase A (Zomanbio, Beijing, China) at 37°C. Total RNA was isolated using TRIzol (Invitrogen) with DNase I treatment. The cDNA library was total RNA directly reverse transcribed using an MMLV Reverse Transcriptase first-Strand cDNA Synthesis Kit with a random hexamer primer (Invitrogen). The negative control was total RNA not treated with MMLV Reverse Transcriptase.
PCR experiments were performed in duplicates using the TransStart FastPfu Fly polymerase (TransGen Biotech, Beijing, China). PCR program settings were 95°C for 5 min (initial denaturation); 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, for 45 cycles. PCR products were visualized after electrophoresis in 2% ExRed-stained (Zomanbio, Beijing, China) agarose gel, and purified through the V-ELUTE Gel Mini Purification Kit (Zomanbio, Beijing, China). The pBLUE-T plasmid was constructed using the pBLUE-T fast cloning kit (Zomanbio, Beijing, China), and Sanger sequencing was performed.