The cDNA was then divided and used for PCR amplification of antiviral protein and cytokine expression. Real-time RT-PCR assays were performed on LightCycler

System 480 (Roche Molecular Diagnostics, Mannhein, Germany) using SYBR Green PCR Master Mix (Roche Molecular Diagnostics). MxA, PKR, OAS, SLPI, IFN-α, IFN-β, IFN-λ, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were amplified using specific primers purchased from Operon (Ebersberg, Germany). The primer sequences are shown below. MxA (5′-GCTACACACCGTGACGGATATGG-3′/5′-CGAGCTGG ATTGGAAAGCCC-3′), PKR (5′-GCCTTTTCATC KU-60019 concentration CAAATGG AATTC-3′/5′-GAAATC TGTTCTGGGCTCATG-3′), OAS (5′-CATCCGCCTAGTCAAGCACTG-3′/5′-CCACCACCCAAGTTT CCTGTAG-3′), SLPI (5′-TTCCCCTGTGAAAGCTTGATTC-3′/5′-GATATCAGTGGTGGAGCCAAGTC-3′), IFN-α (5′-GGATGAGACCCTCCTAGACAAAT-3′/5′-ATGATTTCTGCTCTGACAACCTC-3′), IFN-β (5′-GATTCATCTAGCACTGGCTGG-3′/5′-CTTCAGGTAATGCAGAATCC-3′),

SCH 900776 solubility dmso IFN-λ (5′-GGACGCCTTGGAAGAGTCACT-3′/5′-AGAAGCCTCAGGTCCCAATTC-3′), and GAPDH (5′-GAAGGCTGGGGCTCATTT-3′/5′-CAGGAGGCATTGCTGATGAT-3′). Amplification conditions, sequences, and concentrations of the primers were similar to those of RT-PCR. After 45 reaction cycles, the melting curve analysis was performed at 95°C for 5 s, 65°C for 1 min, and heating to 97°C using a ramp rate of 0.11°C/sec with continuous monitoring of fluorescence. The melting peak generated represented the specific amplified product. All samples had only a single peak, indicating a pure product and no primer/dimer formation. Amplicons of a single band with the expected sizes were also confirmed in all reactions by agarose gel electrophoresis. The amplification efficiencies were high (close to 100%) when multiple standard curves were performed using serial mRNA dilutions. For periodontal tissue specimens, the relative mRNA expression of antiviral proteins and cytokines was normalized to corresponding GAPDH for each sample, using the formula = 2−ΔCT, where ΔCT

= CT-geneX-CT-GAPDH. The relative quantification of mRNA expression in Fossariinae periodontitis tissues was presented as the mean fold increase ± SEM, using the mean value obtained from the healthy tissue as a reference (relative quantification = 1). For HGEC culture, fold differences in mRNA expression levels of antiviral proteins and cytokines between sample A and sample B was calculated using the ΔΔCT method [[47]]. Levels of gene of interest were normalized to corresponding GAPDH for each sample, and the fold increase between sample A and sample B was calculated as follows: Fold increase = 2−ΔΔCT, where The excised periodontal tissues were immediately washed in normal saline solution, placed in the optimum cutting temperature embedding compound, snap-frozen in liquid nitrogen, and stored at −80°C. Single immunohistochemical staining was performed via Polymer/HRP and DAB+ chromagen system (DAKO EnVision™ G/2 Doublestain System, Glostrup, Denmark) on the frozen sections.