Pcr amplifier

The total RNA was extracted by TRIzoL rapid extraction method as above. RNA was reverse transcribed using 5 × RT Master Mix (TOYOBO, Japan) and 2 μg RNA template. The amplification procedure was used as follow: 37°C for 15 min; 50°C for 5 min and 98°C for 5 min in PCR Amplifier (Biometra, Germany). RT‐PCR was performed using 25 ng of cDNA samples using 5 mmol/L of each primer (Table 2) and 5 μl of Brilliant SYBR Green QPCR Master Mix (Takara, Japan) in a Light Cycler apparatus (Roche, Switzerland). The amplification procedure was used: 95°C for 5 min; 40 cycles at 95°C for 5 s; 60 °C for 30 s. All values were normalized to the GAPDH housekeeping gene and relative expressed as fold change using the formula 2^−△△Ct. The primers for RT‐PCR are listed in Table 2.

The PCR amplifier is from Biometra, Germany; the low-temperature ultracentrifuge is from Eppendorf, Germany; the gel imaging analyzer is from AAB, the United States (−20°C low-temperature refrigerator, −80°C low-temperature refrigerator, electric thermostatic water bath, electronic analytical balance, biological). The safety cabinets are all from the Shanghai Lishen Scientific Instrument Company.