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.