4.12. Quantitative Real-Time PCR

The expression levels of MMP2 (forward primer 5’-TTC TTC GCA GGG AAT GAG-3’; reverse primer 5’-ACG ACA GCA TCC AGG TTA T-3’) and MMP9 (forward primer 5’-AAA TGT GGG TGT ACA CAG GC-3’; reverse primer 5’-TTC ACC CGG TTG TGG AAA CT-3’) [78] were analyzed by quantitative real time PCR (qPCR). Reactions were performed by Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific, MA, USA) in a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s recommendations, using 100 ng cDNA and primers specific for each gene and ACTB1 as internal control for normalization. Each sample was tested in triplicate and relative gene expression was analyzed by 2−ΔΔCt method [79].