Real-time quantitative PCR analysis

Total RNA was extracted from HAFC with TRIzol reagent (Life Technologies), was added with 1 μl SUPERase•In™ RNase Inhibitor (Thermo Scientific) and was assessed for quality by the OD260/OD280 ratio. Each RNA sample was quantified with One Step RT-PCT kit (Takara). And the RT-qPCR assay was performed for the reverse transcription firstly (at 42°C for 5 min, and then at 95°C for 10 s) and then for the PCR reaction (at 95°C for 5 s and at 60°C for 20 s, 40 cycles) using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). The specificity of amplified products was determined by melting curve analysis. The relative expression levels of mRNA were calculated with the 2^−ΔΔCt method [34] and were presented as the relative quantitative value normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primers used in the study were as follows: MMP-1 forward (5′-gcc ttc caa ctc tgg agt aa-3′) and reverse (5′-ttg acc ctc aga gac ctt gg-3′); MMP-3 forward (5′- tgc caa aag atg ctg ttg att-3′) and reverse (5′-gag tca cct ctt ccc aga ct-3′); MMP-9 forward (5′-agc tgt att tgt tca agg atg-3′) and reverse (5′-aag ggg ccc tgc ggc cgg ctc-3′); tissue inhibitor of matrix metalloprotease-1 (TIMP-1) forward (5′-gtg ttt ccc tgt tta tcc atc-3′) and reverse (5′-cgt cca caa gca atg agt gc-3′); TLR-2 forward (5′-ttg tga ccg caa tgg tat ctg-3′) and reverse (5′-gcc ctg agg gaa tgg agt tta-3′); TLR-4 forward (5′-tgg tgt ccc agc act tca tc-3′) and reverse (5′-gcc agg tct gag caa tct cat a-3′); TLR-6 forward (5′-cca gga aaa agg gag act tct c-3′) and reverse (5′-tct aca atg ggg tgc aca gtg-3′); RAGE forward (5′-gag cca gaa ggt gga gca gta-3′) and reverse (5′-gca agg gca cac cat cct-3′); NF-κB forward (5′-tgc acc acc aac tgc tta gc-3′) and reverse (5′-tct tct ggg tgg cag tga tg-3′); IκBα forward (5′-acc tgg tgt cac tcc tgt tga-3′) and reverse (5′-ctg ctg ctg tat ccg ggt g-3′); GAPDH forward (5′-tgc acc acc aac tgc tta gc-3′) and reverse (5′-tct tct ggg tgg cag tga tg-3′).