RNA isolation and quantitative real-time PCR

Total RNA was extracted with an RNeasy kit (Fermentas Life Sciences) in accordance with the manufacturer's protocol. Real-time PCR was performed in 20-μL reactions containing 2 μL cDNA, 0.3 μL of each primer, 7 μL ddH₂O, 0.4 μL ROX reference dye, and 10 μL fluorescent SYBR Green (TaKaRa Bio, Inc.). Amplification was carried out in 96-well optical plates on a 7300 Real-Time PCR System (Applied Biosystems) at 95°C for 30 s, followed by 45 cycles of 95°C for 5 s and 60°C for 60 s. Each sample was analyzed in triplicate.