Hiscript 3 rt supermix for quantitative pcr qpcr

To amplify chGSK3β, primers chGSK3β F and chGSK3β R (Table 1) were designed based on the predicted chGSK3β sequence in GenBank XM_040660411.2. Total RNA was extracted from HD11 cells by the method of the FastPure cell/tissue total RNA isolation kit v2 (Vazyme, Nanjing, China) and reverse transcribed into cDNA using the HiScript III RT SuperMix for quantitative PCR (qPCR) (Vazyme), and PCR was performed to amplify the target sequence. The pCAGGS-chGSK3β-Flag vector was constructed by inserting the PCR product of chGSK3β into a pCAGGS empty vector digested by XhoI and NotI.

Human embryonic kidney HEK293 cells were firstly seeded in six wells in 24-well plates (4 × 10⁵ cells per well). They were cultured in high-glucose DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO₂ in humidified atmosphere. According to manufacturer’s instructions of PEI Transfection Reagent, the NAT10-overexpressing plasmids were transfected into HEK293 cells in the experimental group, and the control group cells were transfected with no-load plasmids. After 48 h, the cells were exposed to Actinomycin D (5 μg/ml, Aladdin, A113142) to block RNA synthesis as previously described (24 (link)). In addition, HEK293 cells were then harvested at 0, 2, and 12 h, respectively. The total RNA of cells was then extracted and reversely transcribed using the RNeasy Micro Kit (Qiagen, 74004) and HiScript III RT SuperMix for Quantitative PCR (qPCR) (Vazyme, R323-01) according to the manufacturer’s instructions. RealStar Green Power Mixture (2×) (Genstar, A311-101) was used to carry out qPCR on the Roche LightCycler 480 II (Roche Diagnostics, Germany). The expression levels of OGA mRNA in the experimental group and the control group at 0 h were normalized to 1, and the relative expression levels at 2 and 12 h were calculated respectively based on 2^−ΔΔCT method. In addition, the primers are displayed in Table 1.