Tb green qpcr reagent

About 5 × 10⁸ cells in each of four groups were collected, 500 μl TRIzol (R0016, Beyotime) lysate protein nucleic acid complex was added, and 100 μl chloroform was mixed to extract RNA. After centrifugation at 12000 RPM, 4°C, for 10 min, the upper colorless aqueous phase was collected, 200 μl isopropanol was added to precipitate RNA, and 75% of the cells were washed. The extracted RNA was retrotranscribed into a cDNA template with a reverse transcription reagent (RR037Q, Takara). Then, a real-time fluorescent quantitative polymerase chain reaction (Applied Biosystems, ABI 7500) was conducted with TB Green qPCR reagent (RR82LR, Takara). The Ct value of genes to be examined was calibrated using the Ct value of the internal reference gene GAPDH, and the amplification multiple of genes relative to the internal reference genes was calculated according to the 2^−∆∆Ct formula. Amplification conditions: the first stage was kept at 95°C for 30 s; the second stage was 95°C for 5 s and 60°C for 30 s, for 40 cycles; and the third stage was maintained at 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s.

After sacrificing mice with carbon dioxide, the liver was collected and washed for residual blood with 0.9% saline and stored at −80°C. 30 mg tissues was taken, fully broken on the ice in homogenate, with 1 ml TRIzol RNA separation reagent (Thermo Fisher, 10296010). After resting 5 min, the tissues were centrifuged at 12000 rpm for 15 min, and the upper clarification aqueous phase was taken. RNA was precipitated with isopropanol and washed with 75% ethanol solution. After the precipitation was air-dried, RNA concentration was determined with a microplate analyzer. After recording the RNA reversal to a cDNA template with the Reversal Reagent (Takara, RR037Q), qRT-PCR (Applied Biosystems ABI 7500) was performed with TB Green qPCR reagent (Takara, RR82LR). Amplification conditions: (1) Phase I, 95°C for 30 s; (2) Phase II, 95°C for 5 s, 60°C for 30 s, for 40 cycles; (3) Phase III, 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. The amplification multiple of genes relative to the internal reference was calculated by 2^{−∆∆ Ct} formula according to Ct value. The upstream and downstream primer sequences are shown in Table 1.