RNA was extracted as described above and concentrated using an RNA Clean and Concentration kit (Zymo Research, Irvine, CA, USA). All samples were normalized with nuclease free water to a concentration of 50 ng/µL. 250 µg of RNA was synthesized into cDNA using Superscript III reverse transcriptase (Invitrogen|Thermo Fisher Scientific, Waltham, MA, USA) as previously described [19 (link)]. To identify any genomic DNA contamination, non-template controls of each RNA sample were also prepared and verified by reverse transcriptase PCR (RT-PCR) using GAPDH primers [42 (link)]. All contaminated samples were discarded. Quantitative reverse transcriptase PCR (qRT-PCR) was performed using Sybr green reagent (Applied Biosystems|Thermo Fisher Scientific, Waltham, MA, USA) using primers for CCL20, CERS2, CSF2, ICAM-1, IL-1α, IL-1β, IL-6, IL-8, MMP1, MMP9, TNFα [42 (link),43 (link),44 (link),45 (link),46 (link),47 (link),48 (link),49 (link),50 (link),51 (link),52 (link)]. All gene reactions were normalized to GAPDH [42 (link)], and analyzed using the ΔΔCT method. All experiments were performedat least three independent times.
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