Total RNA was extracted using an RNAEasy kit according to the manufacturer’s instructions (Bioecon Biotec Co., Shanghai, People’s Republic of China). RNA purity was checked by measuring the OD260/280 of RNA samples. cDNA was synthesized through reverse transcription using M-MLV reverse transcriptase and oligo(dT) primer. The gene expression levels of SIRT1, TNF-α, IL-6, IL-1β, and iNOS were detected by real-time PCR. PCR amplification was performed in a 96-well plate in triplicate. Each reaction well consisted of 10 µL of 2× SYBR Green PCR Master mix, 0.5 µL forward and reverse primers, and 1 µL of template cDNA to obtain a final volume of 20 µL. The PCR analysis was performed using a Mastercycler ep realplex apparatus (Eppendorf, Germany). After an initial denaturation step for 3 minutes at 95°C, the conditions for cycling were 39 cycles of 30 seconds at 95°C, 30 seconds at 60°C, and 15 seconds at 72°C. The primer sets for SIRT1, TNF-α, IL-6, IL-1β, iNOS, and GAPDH were purchased from Dingguo Changsheng Biotechnology Co. (Beijing, People’s Republic of China) and are listed in Table 1 .
The relative expression level of each target gene was normalized against the GAPDH control using the 2−ΔΔCt method and further compared with its own control. All samples were studied in independent triplicate experiments.
The relative expression level of each target gene was normalized against the GAPDH control using the 2−ΔΔCt method and further compared with its own control. All samples were studied in independent triplicate experiments.