Quantitative real-time PCR assay
Peripheral blood mononuclear cells were isolated from peripheral blood
using density gradient centrifugation. Then, total RNA were extracted
and purified from peripheral blood mononuclear cells by
TRIzolTM LS
reagent. Total RNA was reverse translated as complementary DNA after the
degradation of mixed genomic DNA using PrimeScriptTMRT reagent Kit with gDNA Eraser (Takara Bio Inc., Japan). SYBR Green kit
(Takara Bio Inc., Japan) was used in quantitative real-time PCR process
based on QuantStudioTM 7 Flex (Life Technologies,
USA). The expression data of ERAP1 was normalized to internal
reference β-actin, and the relative expression level of ERAP1 was
calculated by comparative 2-ΔΔCt method. Primers
sequence of ERAP1 and
β-actin
were listed as following: β-actin, forward: 5’-CTCCATCCTGGCCTCGCTGT-,
reverse: 5’-GCTGTCACCTTCACCGTTCC-; ERAP1, forward:
5’-TTTGAACTTGGCTCATCTTCC-, reverse: 5’-AATTGTCTGTTGGACACAACG .