Fibroblast gene expression analysis

Differences in mRNA expression of IL-1β, TNF-α, GAL-1, GAL-3, MMP-3, collagen type I, collagen type III and elastin genes were analysed by qPCR in primary human fibroblasts (SK0355 and SK0235) exposed or not to activated U937 CM treated with HA, CTL and their mixture for 4, 10 and 24 h.
Total RNA was extracted using TRIzol (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA quality was monitored using Nanodrop 2000c spectrophotometer (Thermo Scientific) by measuring absorbance at 260/280 nm. Total RNA was incubated with DNAse I (ThermoFisher) for 15 min to remove DNA from the samples. In total, 500 ng of total RNA was reversely transcribed using Oligo-dT and Superscript II (Life Technologies, Carlsbad, CA, USA) and qPCR was carried out using Xpert quick SYBR Green (GRISP, Portugal) on a Rotor-Gene RG -3000A (QIAGEN, Germany) [20 (link),21 (link)]. Primers used for q-PCR analysis are listed in Table 1. Gene expression was assessed using the 2ΔCt method, in which ΔCt = Ct peptidylprolyl isomerase A (PPIA)-Ct target gene.