Real-time polymerase chain reaction

Complementary DNA and primers were added to 15 μL total reaction volume of Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Polymerase chain reactions (PCRs) were then performed using the StepOne Real-Time PCR system (Applied Biosystems). Conditions were set to the following parameters: 10 minutes at 95°C, followed by 40 cycles each of 15 seconds at 95°C and 1 minute at 60°C to 64°C. The primer sets are shown in Supplemental Table 1. L19 was used as a control for reaction efficiency and variations in concentrations of mRNA in the original real-time PCR.