Quantitative real-time PCR

Tissues (50 mg) or cells (1×10⁷) were trypsinized and lysed with 1 mL TRIzol. After lysis, 200 μL of chloroform was added to the cleavage product, which was placed at room temperature after 20 minutes. RNA was transferred to the new Eppendorf (EP) tube, and 1 mL of isopropanol was added to the RNA and centrifuged at 10 000 g for 10 minutes. One milliliter of 75% ethanol was used to wash RNA, then centrifuged at 10 000g for 10 minutes, and dissolved in 50 μL of DEPC water to obtain RNA. A PrimeScript RT Reagent Kit (R&D Systems, MN, USA) was used to generate RNA by reverse transcriptase cDNA. The PCR system (10 μL) included 2.0 μL of PrimeScript Buffer, 0.5 μL of PrimeScript RT Enzyme, 0.5 μL of Oligo dT Primer (50 μM), 0.5 μL of random 6 mers (100 μM), 500 ng of RNA, and ddH2O to a 10-μL volume. The reverse transcription conditions were as follows: initial denaturation at 95°C for 5 minutes; and 40 cycles of 95°C for 15 second and 60°C for 1 minute. The data were preserved.