RNA extraction and quantitative RT-PCR
Total mRNA was purified from peripheral blood cells using Trizol
(Invitrogen) and quantified by an ultraviolet spectrophotometer (Thermo
Fisher Scientific, USA) [20]. 1μg RNA was reverse transcribed into
cDNA using Reverse Transcriptase Kit (Qiagen, Netherlands) accordance to
the manufacturer’s instructions [21]. Then, quantitative RT-PCR was
executed using SYBR® Premix Ex TaqTMII system (TaKaRa, Japan) with the CFX96 TouchTMReal-Time PCR Detection System (Bio‐Rad, USA).
1μl of the reverse-transcript was
added to a 30 μl PCR mixture for 40 cycles. Each cycle included 93℃ for
30s and 54℃ for 60s. By the comparison between the copy numbers of
target gene and β-actin, the normalization of mRNA expression data for
sample-to-sample variability in RNA input, RNA quality and reverse
transcription efficiency was completed. Primer sequences were described
in Table 1.