RNA was extracted from RAW264.7 cells using the GenElute mammalian total RNA Miniprep kit (Sigma-Aldrich, St. Louis, MO, United States) with on-column genomic DNA digestion in accordance with the manufacturer’s instructions. For gene expression analysis of antioxidant enzymes, RNA was extracted at 1 day after Bach1 inhibitors treatment, and the gene expressions of Hmox1 and Nqo1 was analyzed because these anti-oxidant enzymes were reported to relate osteoclastogenesis (Zwerina et al., 2005 (link); Sakamoto et al., 2012 (link); Hyeon et al., 2013 (link)). For gene expression analysis of osteoclast markers, RNA was extracted at 4 days after RANKL stimulation, and the gene expressions of Atp6v0d2, Cathepsin K, Matrix metalloproteinase 9, Trap, Dcstamp, and Oscar were analyzed. RNA (500 ng) was reverse transcribed with iScript cDNA-Supermix (Bio-Rad Laboratories, Hercules, CA, United States).
Real-time RT-PCR was performed with SsoFast EvaGreen-Supermix (Bio-Rad Laboratories). PCR primers used in the experiments were from PrimerBank and are previously described (Kanzaki et al., 2013 (link)). Fold changes of gene of interest were calculated by using the Δ-Δ Ct method with Ribosomal protein S18 as reference gene. Data shown are representative of three independent experiments performed in triplicate.
Real-time RT-PCR was performed with SsoFast EvaGreen-Supermix (Bio-Rad Laboratories). PCR primers used in the experiments were from PrimerBank and are previously described (Kanzaki et al., 2013 (link)). Fold changes of gene of interest were calculated by using the Δ-Δ Ct method with Ribosomal protein S18 as reference gene. Data shown are representative of three independent experiments performed in triplicate.
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