Power eva qpcr supermix kit

Total RNA was isolated from liver tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, 15596018). Reverse transcription was performed as following: 0.5 μg of total RNA, 4 μl of 2.5 mM dNTP, and 2 μl of 15 μM random primers (New England Biolabs, Ipswich, MA, S1254S) were mixed at a volume of 16 μl, and incubated at 70 °C for 5 min. Then, a 4 μl cocktail containing 25 units of Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase (New England Biolabs, Ipswich, MA, M0253S), ten units of RNasin Plus (Promega, Madison, WI, N261B) and 2 μl of 10x M-MuLV Reverse Transcriptase Reaction Buffer (New England Biolabs, Ipswich, MA, B0253S) was added, and samples were incubated at 42 °C for 1 h and then at 95 °C for 5 min. The cDNA was diluted and used for real-time quantitative PCR (qPCR) using the Power Eva qPCR SuperMix Kit (Biochain, Newark, CA, K5057400), following manufacturer’s protocol. The qPCR was performed on the StepOne PCR System (Applied Biosystems, Foster City, CA) and analyzed with the ∆∆-Ct method, as supplied by the manufacturer (Applied Biosystems, Foster City, CA). Rpl19 gene expression was used for internal normalization. Primers are listed in Supplementary Table 1.