Qrt pcr

Ten whole blood samples from CAD patients were collected from the Second Hospital of Hebei Medical University, 10 whole blood samples from the health check-up population were collected from the Great Wall Physical Examination Center, and total blood RNA from the population was extracted using the RNAprep Pure Hi-Blood Kit.
The mouse cardiomyocyte line HL1 was cultured in MEM containing 10% fetal bovine serum (FBS) at 37°C in a 1% O2 incubator. The coronary artery disease group was given 20 ng/mL TNFα treatment. Total cellular RNA was extracted using TRIpure (Aidlab, CN). The procedure was performed according to the manufacturer’s instructions.
RNA quality and concentration were assessed using a NanoDrop 2000 (Thermo Fisher Scientific, United States). Then, we used a cDNA synthesis kit (Thermo Fisher Scientific, #K1622) to obtaine cDNA by reverse transcription, and analyzed by using qRT‒PCR (Tiangen, FP205). Finally, mRNA expression was normalized to the GAPDH gene.

The human normal hepatocyte line LO2 and HCC cell lines HepG2 and HL-7721 were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, USA) containing 10% fetal bovine serum (FBS, zeta life, USA) in a 5% CO₂ incubator at 37°C. Total cellular RNA was extracted using TRIpure (Aidlab, Beijing, CN). The procedure was performed according to the manufacturer’s instructions. Total RNA was extracted from liver tissue and hepatocyte lines with TRIZOL (TIANGEN, Beijing, CN) reagent. NanoDrop-2000 (Thermo Fisher Scientific, Waltham, MA, USA) was used to assess the RNA quality and concentration. cDNA was then obtained by reverse transcription with a cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA), and qRT-PCR (TIANGEN, FP205, GER) was used for analysis. Finally, the mRNA expression was normalized with the GAPDH gene. The specificity of the qRT-PCR products was evaluated by observing whether the melting curve was a single peak. Three replicate wells were set up for each reaction, and those with Cq values not exceeding 0.5 between replicate wells were used for data analysis, in which the mean Cq values were calculated.

RNA was extracted from cultured cells with TRIzol reagent (Takara, China). Total RNA was reverse-transcribed into cDNA according to the instructions of the reverse transcription kit (TIANGEN, China). An appropriate amount of cDNA was used as the template for PCR and amplified according to the qRT-PCR specifications (TIANGEN, China). Finally, the gene expression was analyzed with GAPDH and U6 as internal references, and relative expression levels were calculated by using the 2^−△△Ct method. PCR was performed using an ABI 7500 instrument (Applied Biosystems, USA). The primers used for real-time PCR analysis are listed in Table 1.