Moloney murine leukemia virus rt

Horse skeletal muscle tissue (~50 to 100 g) was crushed using a mortar, and ground muscle tissue was then dissolved using 1 mL TRIzol (Invitrogen, Karlsruhe, Germany). Next, 200 μL of chloroform was added to remove cells from the organic solvent, the mixture was shaken for 10 s, maintained at 4°C for 5 min, and centrifuged at 4°C for 15 min. The supernatant was removed and added to a new test tube, mixed with an equal amount of isopropanol, and maintained at 4°C for 15 min to collect RNA pellets. Isopropanol was removed from the solution via centrifugation at 4°C for 15 min, and the sample was then sterilized with 85% ethanol and dissolved in RNase-free water. The purity of the extracted RNA was confirmed by measuring absorbance at 230 nm and 260 nm using a spectrophotometer (ND-100, Nanodrop Technologies Inc., Wilmington, DE, USA), and only RNA samples with purity (optical density value of 230 nm/260 nm) measurements greater than 1.8 were selected and stored at −70°C until the experiment was carried out.
To synthesize cDNA, 1 μg of RNA and 1 μL each of oligo-dT (Invitrogen, USA) and RNase-free water were added. RNA was denatured at 80°C for 3 min, and cDNA was synthesized using 4 μL of 5×RT (reverse transcription) buffer, 5 μL of 2 mM dNTPs, 0.5 μL of RNase inhibitor (Promega, Madison, WI, USA), and 1 μL of moloney-murine leukemia virus RT (Promega, USA).

Total RNA from INS-1E cells were isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the instructions of the manufacturer. RNAs were transcribed to cDNA at 42°C for 1 hour in a 25 μl cocktail containing 5× reverse transcriptase (RT) buffer, 10 mM dNTPs (200 units), Moloney murine leukemia virus RT (Promega, Madison, WI, USA), and 100 pm oligo-dT primer. After the termination of cDNA synthesis, the mRNA levels of target genes were determined by quantitative RT-PCR as described previously [24 (link)]. The relative amounts of the mRNA levels of the target genes were normalized to β-actin, and the relative difference in mRNA levels was calculated by the 2^-ΔΔCt method. The sequences of primers and reaction conditions used are listed in Table S1 and Table S2.