Real-Time RT-PCR for Gene Expression

For real-time RT-PCR, we used a TB Green Premix Ex Taq II (Takara Bio, Shiga, Japan). For each 2 μL of the cDNA sample, 1.6 μL of primer (Forward and Reverse, 0.8 μL each) (Table 1) (Takara Bio) was added, and 8.5 μL of RNase free water, 12.5 μL of TB Green Premix Ex Taq II and 0.4 μL of Rox Reference Dye was added to make a total of 25 μL. The expression level of cDNA was measured using an Applied Biosystems^® 7500 real-time PCR system (Thermo Fisher Scientific). The holding stage was at 50 °C for 2 min and 95 °C for 10 min. The cycling stage for 40 cycles was at 95 °C for 15 s, and 60 °C for 1 min. The melt curve stage was at 95 °C for 15 s, 60 °C for 1 min, 95 °C for 30 s, and 60 °C for 15 s. The analysis of the gene expression level of each sample was carried out in comparison with the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which is a housekeeping gene. The expression levels of DUSP1 were calculated as correction values divided by the expression level of GAPDH. The primer sequences of each gene used in this study were the same as in the previous report [7 (link)].