Rr037q

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.

The TRIzol method (R0016, Beyotime, China) was applied to extract RNA. For reverse transcription of RNA into cDNA (RR037Q, TAKARA, Japan), the reaction system was as follows: a final volume (10 μl) reaction system containing, 5× PrimeScript RT Master Mix (2 μl), total RNA (2 μl), and RNase-free ddH₂O (6 μl). The reaction conditions were set as follows: 37°C for 15 min, 85°C for 5 s, and 4°C for 5 min. The reaction volume for real-time fluorescent quantitative PCR (RR086A, TAKARA, Japan) was 25 μl comprising 2× SYBR Premix (12.5 μl), forward and reverse primers (1 μl each), cDNA (2 μl), and ddH₂O (8.5 μl). The reaction conditions were as follows: 95°C for 30 s, 95°C for 5 s, 60°C for 30 s (40 cycles). Each sample had four replicate wells, with tubulin as the internal control. The 2^−ΔΔ_t method was used to calculate the relative expression levels. The reference primer sequences were, forward primer: CGG TTC ATG GAA ACC TCA CT, and reverse primer CAT GCT GGC TCC GGT ATA AT.