2.6 RNA isolation and quantitative RT-PCR analysis
Complete RNA was extracted from dorsal skin tissue using TRIzol (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (ThermoScientific, MA, USA) following the manufacturer’s instructions. Quantitative RT-PCR analysis of gene expression was done using FastStart Universal SYBR Green Master (Roche, Basel, Switzerland). The levels of different target genes normalized against GAPDH were calculated using 2-ΔΔCt methods. Primer sequences (5′ to 3′) were as follows: murine GAPDH forward: CTCGTCCCGTAGACAAAATGGT, reverse, GAGGTCAATGAAGGGGTCGTT; murine IL-1β forward: GAAATGCCACCTTTTGACAGTG, reverse, TGGATGCTCTCATCAGGACAG; murine IFN-α forward: GATGCCCTGCTGGCTGTGA, reverse, CTTCTGCTCTGACCACCTCCC; murine IL-6 forward: CTGCAAGAGACTTCCATCCAG, reverse, AGTGGTATAGACAGGTCTGTTGG; murine IFN-γ forward: TCGGTAACTGACTTGAATGTCCA, reverse, TCGCTTCCCTGTTTTAGCTGC; murine Il4 forward: GGTCTCAACCCCCAGCTAGT, reverse, GCCGATGATCTCTCTCAAGTGAT; murine Il13 forward: TGAGCAACATCACACAAGACC, reverse, GGCCTTGCGGTTACAGAGG.