Real-time RT-PCR

Total cellular RNA of whole vaginas harvested from the mice was isolated with RNeasy Mini Kits (Qiagen, Valencia, CA), and was transcribed to cDNA using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Real-time RT-PCR was performed on an iCycler iQ detection system (Bio-Rad) using Sybergreen (Bio-Rad) for real-time monitoring of the PCR. The primers used were as follows: IFNγ, 5’-TAC TGC CAC GGC ACA GTC ATT GAA-3’, 5’-GCA GCG ACT CCT TTT CCG CTT CCT-3’; IL-4, 5’-GAA GCC CTA CAG ACG AGC TCA-3’, 5’-ACA GGA GAA GGG ACG CCAT-3’; IL-17A, 5’-TCA GGG TCG AGA AGA TGC TG-3’, 5’-TTT TCA TTG TGG AGG GCA GA-3’; β-actin, 5’-CCT AAG GCC AAC CGT GAA AAG-3’, 5’-GAG GCA TAC AGG GAC AGC ACA-3’. Relative quantification of target genes was analyzed based on the threshold cycle (Ct) determined by Bio-Rad iQ5 optical system software.