Quantitative RT-PCR Analysis of Vaginal Gene Expression

mRNA from vaginal tissue was extracted using Trizol after homogenization as previously described (45 (link)). First strand cDNA was synthesized by reverse transcription, according to the manufacturer’s protocol (BioRad). Then, the first strain cDNA was used for quantitative real-time PCR using IQ SYBR Green (Invitrogen) on the MyiQ real-time detection system (BioRad) following the manufacturer’s protocol. The forward and reverse primers for β-actin sense-ACTGGGACGACATGGAGAAG, antisense-GGGGTGTTGAAGGTCTCAAA, TLR-7 sense-CCACAGGCTCACCCATACTTC, antisense-GGGATGTCCTAGCTGGTGACA, CXCR-9 sense-CAAATCCCTCAAAGACCTCAAAC, antisense-GATCTCCGTTCTTCAGTGTAGC, CXCR-10 sense-TCATCCCTGCGAGCCTAT, antisense-CTTGATGGTCTTAGATTCCGGAT, IFN-γ sense-ACAATGAACGCTACACACTGCAT, antisense-TGGCAGTAACAGCCAGAAACA, and β-Actin sense-ACTGGGACGACATGGAGAAG, antisense-GGGGTGTTGAAGGTCTCAAA(23 ). Gene expression levels were normalized to β-Actin housekeeping gene, and data were represented as fold differences by the 2^−ΔΔCt method, where ΔCt = Ct_{target gene} − Ct_β-Actin and ΔΔCt = ΔCt_induced − ΔCt_reference, as previous mentioned (46 (link)).