Quantitative Assessment of Antioxidant mRNA

SW1353 cells (4 × 10⁵) were seeded in a 6-cm dish and treated with MIA and/or zinc for 24 h, and then the total RNA was extracted by using REzol reagent (Protech, Taipei, Taiwan) according to the manufacturer’s instructions, as previously described [19 (link)]. Each experiment was carried out in triplicate. The complementary DNA (cDNA) was synthesized from random primed reverse transcription from 2 μg of total RNA using M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA) according to the manufacturer’s instructions. Real-time PCR was performed on a MiniOpticon^TM Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) using iQ^TM SYBR^® Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) as previously described [20 (link)], was used to confirm the results of real-time PCR. The mRNAs encoding GCLC, GCLM, IL-10, and IL-1β were measured using real-time PCR, with RPS18 mRNA as the housekeeping gene. The primers and amplified products of each gene used in the present study are shown in Table 1. The cycle threshold (C_t) value of the target gene was normalized to RPS18. The data were calculated and expressed as 2^−ΔΔCt [21 (link)] using MJ Opticon Monitor Analysis software version 3.1 (Bio-Rad Laboratories, Hercules, CA, USA).