Chamq universal sybr qpcr maser mix

mRNA was quantified with qRT-PCR according to a protocol reported elsewhere [19 (link)]. Total RNA was extracted from the cells or tissues using the RNA Extraction Reagent (R401-01, Vazyme Biotech, China). RNA was quantified with SpectraMax i3× (Molecular Devices) and mRNA (100 ng/μl) was transcribed into cDNA using the HiScript® II Q RT SuperMix for qPCR (+g DNA wiper) (R223, Vazyme Biotech, China). mRNA was quantified with ChamQ™ Universal SYBR qPCR Maser Mix (Q711,Vazyme Biotech) on LightCycle 480 II (Roche). The result was normalized with the Gapdh signal. The primer sequences are listed in Table 1. The relative expression level of Mecr mRNA was calculated by 2^−ΔΔC_t method.

mRNA of G6pas, Fbp1, Pck1 (Pepck1) was quantified with qRT-PCR according to a published protocol (19 (link)). Total RNA was extracted from the liver tissues and primary hepatocyte using the RNA Extraction Reagent (R401-01, Vazyme Biotech, China), which was quantified by SpectraMax i3× (Molecular Devices). mRNA was transcribed into cDNA using the HiScript^® II Q RT SuperMix for qPCR (+g DNA wiper) (R223-01, Vazyme Biotech, China), which was quantified with ChamQ™ Universal SYBR qPCR Maser Mix (Q711-02, Vazyme Biotech) on Light Cycle 480II (Roche). The primer sequences were listed as follows: G6pase forward, 5’-GCCTTCTATGTCCTCTTTCCC-3’ and reverse, 5’-GCGTTGTCCAAACAGAATCC-3’; Fbp1 forward, 5’-ATGGATTGTGGTGTCAACTG-3’ and reverse, 5’-CTCATTAAGGCTGTAGATGTTACC-3’; Pck1 forward, 5’-GGAAGAACAAGGAGTGGAGAC-3’ and reverse, 5’-CAGGCAGGGTCAATAATGGG-3’; P2Y1 forward, 5’-GACTGACTGGATCTTCGGGGA-3’ and reverse 5’-CCACCACAATGAGCCACACC-3’; P2Y12 forward, 5’-CCACTAACTAGTATTCCCGGAGAC-3’ and reverse, 5’-GATGAGCCCAGCAAAGAACA-3’; P2Y13 forward, 5’-GCCTTTCAAAATCCTTTCCGA-3’ and reverse, 5’-TGTTTTTGCGAAAGCCGTCT-3’; 18S forward, 5’-GCCGCTAGAGGTGAAATTCT-3’ and reverse, 5-TCGGAACTACGACGGTATCT-3; The results were normalized as 18S mRNA level.