Total RNA was extracted using Pure Link mRNA mini kit according to the manufacturer’s instructions (Life Technologies, Milan, Italy). A constant amount of RNA (1 μg/sample) was retro-transcribed into complementary DNA (cDNA) and then 1 μl of cDNA/sample was amplified using the following conditions: denaturation 1 minute at 95°C; annealing 30 seconds at 55°C for MMP-2, at 58°C for IL-8 and MMP-9, at 61° for IL-17A and at 60°C for β-Actin, followed by 30 seconds of extension at 72°C. Primers’ sequences were as follows: IL-8 forward 5′-AGGAACCATCTCACTGTGTG-3′, reverse 5′-CCACTCTCAATCACTCTCAG-3′; IL-17A forward 5′-ACTACAACCGATCCACCTCAC-3′, reverse 5′-ACTTTGCCTCCCAGATCACAG-3′; MMP-2 forward 5′-TGACGGAAAGATGTGGTGTG-3′, reverse 5′-GGTGTAGGTGTAAATGGGTG-3′; MMP-9 forward 5′-CGTCTTCCAGTACCGAGAGA-3′, reverse 5′-GCAGGATGTCATAGGTCACG-3′; β-actin forward 5′-AAGATGACCCAGATCATGTTTGAGACC-3′, reverse 5′-AGCCAGTCCAGACGCAGGAT-3′ was used as internal control gene. RNA expression was calculated relative to the housekeeping β -actin gene on the base of the ΔΔCt algorithm.
Abbreviations: IL, interleukin, MMP, matrix metalloproteinase.