Real-time PCR
Total RNA of PBMCs was extracted using TRI Reagent (Molecular Research
Center, Cincinnati, OH, USA) in accordance with the manufacturer’s
instructions. cDNA was synthesized from the total RNA by using
PrimeScript Reverse Transcriptase (Takara Bio, Otsu, Japan) according to
the manufacturer’s instructions. To confirm the mRNA expression ofCOX2 in PBMCs, real-time PCR was performed with the LightCycler
480 System II (Roche Diagnostic, Mannheim, Germany) using SYBR Premix
DimerEraser (Takara Bio) following the manufacturer’s instructions.β-actin (ACTB ) was used as a reference gene. The relative
expression levels were calculated by using the ΔΔCt method, and the
results were indicated as relative change to no treatment group. Primers
were 5’-ACG TTT TCT CGT GAA GCC CT-3’ and 5’-TCT ACC AGA AGG GCG GGA
TA-3’ for COX2 , and 5’-TCT TCC AGC CTT CCT TCC TG-3’ and 5’-ACC
GTG TTG GCG TAG AGG TC-3’ for ACTB .