The materials and methods of Real-Time Quantitative PCR
real-time quantitative PCR
For real-time PCR analysis,0.2 µl of cDNA samples was used in a 25-µl mixture containing 0.2 µM specific primers (Table 1) and 12.5 µl of SYBR Premix Ex Taq (2µ; Takara), using an iQ 5 System (Bio-Rad). A control without cDNA was also included. Amplification was initiated at 95°C for 1 min, followed by 40 cycles at 95°C for 10 s, 55°C for 15 s, and 60°C for 20 s. After amplification, melting-curve analysis was performed. The relative amount of transcript was calculated using the Bio-Rad iQ 5 Optical System software (Bio-Rad) based on the normalized-expression (ΔΔCT, where CT is threshold cycle) method.
