Real-Time PCR for Inflammatory Markers

Total RNA was extracted from cells with REzol reagent (Protech, Taipei, Taiwan) according to the manufacturer’s instructions, as described previously [38 (link)]. 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 directions. Real-time PCR, performed on a MiniOpticonTM Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) using iQTM SYBR^® Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) according to a published procedure [39 (link)], was used to confirm results of real-time PCR. mRNA coding for MMP-1, MMP-3, MMP-9, MMP-13, IL-1β, IL-6, IL-17A, and TNF-α were measured by real-time PCR, with β-actin mRNA being amplified as a housekeeping gene. Primer sequences of targets are listed in Table 1. The cycle threshold (C_t) value of the target gene was corrected by the β-actin. Data were calculated and expressed as ΔΔC_t [40 (link)] by using MJ Opticon Monitor Analysis software version 3.1 (Bio-Rad Laboratories, Hercules, CA, USA).