2.10 RNA isolation, reverse transcription and RT-PCR
Total RNA was extracted from skin lesion using a RNAprep Tissue Kit
(TianGEN, China) and resolved in RNase-free water. The purity of total
RNA was detected by nanodrop 2000 spectrophotometer(Thermo Fisher
Scientific, USA). After reverse-transcript into complementary RNA with
EvoScript Universal cDNA Master (Roche, USA), Real-time PCR was then
performed by using FastStart Universal SYBR Green Master (ROX) (Roche,
USA) on ViiA 7 Dx (Applied Biosystems, USA). Primers were shown as:
TGFβ1, Forward: 5’-CCA TCG ACA TGG AGC TGG TGA AAG G-3’, Reverse: 5’-GCA
GTG AGC ACT GAA GCG AAA GCC C-3’; IL-23 p19, Forward: 5’-TGG GCA TCT GTT
GGG
TCT-3’,
Reverse: 5’-TGG GCA TCT GTT GGG TCT-3’. GAPDH as the control. All
experiments were conducted according to the manufacturer’s instructions.