Quantitative RT-PCR for Gene Expression

RNA extraction from the plasma was performed using the QIAGEN RNeasy Serum/Plasma Maxi Kit (QIAGEN, Germany) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using the PrimeScriptTM RT reagent kit (Takara Bio Inc., Otsu, Japan). Quantitative Reverse Transcription PCR was performed using the SYBR Premix Ex Taq kit (Takara Bio Inc., Otsu, Japan). The procedure is described briefly as follows: 2 μL cDNA, 5 μL TB Green Premix Ex Taq, 0.2 μL PCR forward primer (10 μM), 0.2 μL PCR reverse primer (10 μM), 0.2 μL ROX reference dye, and 2.4 μL nuclease-free water were added in 200 μL tubes. The reaction mixtures were placed in the Roche 480 system (Roche, Rotkreuz, Switzerland), and the reactions were carried out under the following reaction conditions: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s, and annealing and extension at 60°C for 30 s. Each sample was analyzed in triplicate, and the specificity for each PCR reaction was confirmed by the melt curve analyses. The mRNA expression levels were calculated using the 2^-ΔΔCT method with GAPDH as the control [13 (link)].The primer sequences used are shown in Table 1.