Antioxidant Gene Expression Analysis

For antioxidant gene expression analysis, RNA was extracted two days after the treatment of cells with EGCG-GL or gelatin solution, and the expression of Nrf2, heme oxygenase 1 (Hmox1), and glutamate-cysteine ligase (Gclc) were analyzed. RNA was extracted from RAW 264.7 cells using the GenElute mammalian total RNA Miniprep-kit (Sigma-Aldrich, St. Louis, MO, USA) with an on-column genomic DNA digestion. Isolated RNA (500 ng each) was reverse-transcribed with an iScript cDNA-Supermix (Bio-Rad, Hercules, CA, USA). Real-time RT-PCR was performed with SsoFast™ EvaGreen^® Supermix EvaGreen-Supermix (Bio-Rad). PCR primers were from PrimerBank (Boston, MA, USA) and have been described previously [21 (link),23 (link),24 (link)]. Fold changes of gene expression were calculated by using the −∆∆Ct method with ribosomal protein S18 (RPS18) as the reference gene.