Real-time PCR Analysis of Gene Expression

Real-time PCR was performed using the QuantStudio^®5 real-time PCR Design & Analysis system (LightCycler^® 480 II, Roche Diagnostics, USA) with a TB^® Premix Ex Taq™ Kit (Takara Biotechnology Co. Ltd., Dalian, China). The reactions were: 95°C for 30 s (hold stage), followed by 40 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 20 s (PCR stage), then 95°C for 15 s, 60°C for 1 min, 95°C for 15 s (melt-curve stage). A subsequent dissociation stage produced a melting curve to verify the specificity of the amplified products as described in the previous report (37 (link)). The mRNA expression of each gene was normalized to that of β-actin. The fold change relative to the control group was analyzed according to the 2^−ΔΔCT method. The specific sequences of primers are listed in Table 2.