M mlv first strand transcriptase

The gene transcription levels related to oxidative stress, inflammation, mitochondrial dynamics, mitochondrial biogenesis, and mitophagy in liver samples from different treatment groups were analyzed. Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, USA) and then reverse transcribed to cDNA with M-MLV First-Strand Transcriptase (Invitrogen, Shanghai, China). Quantitative RT-PCR was performed on a LightCycle 480 II (Roche Diagnostics, Switzerland) instrument with SYBR Green Real-time PCR Master Mix (Roche Diagnostics, Switzerland). Relevant primers are listed in Table 2. Relative quantification of gene expression was performed as described by Pfaffl (28 (link)).

Total RNA of liver tissue was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and then performed the DNAse treatment. The cDNA was obtained using M-MLV First-Strand Transcriptase (Invitrogen, Shanghai, China). Quantitative RT-PCR was performed on a LightCycler 480 System (Roche, Germany) with SYBR^® Green I Master Mix (Roche, Germany). EF1α was used as housekeeping gene, and PCR were performed using six biological replicates and two technical replicates along with negative controls without reverse transcriptase or template. Melting curves were monitored to confirm the specificity of the amplification reaction. Amplicon identities were confirmed by sequencing. Table S1 shows the primers used for quantitative RT-PCR and amplification efficiencies. The calculation of relative quantification was performed using the Pfaffl’s mathematical model (27 (link)).