一步法Real - time RT - PCR检测HepG2细胞人类白细胞抗原-E基因沉默效率
Detection of interfering efficiency of HLA - E mRNA expression in HepG2 cell using one - step real time RT - PCR method
目的 应用一步法Real - time RT - PCR检测不同载体转染HepG2细胞沉默人类白细胞抗原-E (HLA -E)基因的效率.方法 分别用Gal - PEG - PEI/psiRNA、Gal - PEG - PEI/NC - psiRNA、PEG - PEI/psiRNA、Lipofectamine 2000/psiRNA、裸psiRNA转染HepG2细胞,提取细胞总RNA,一步法Real - time RT - PCR检测HLA -E基因沉默前后mRNA表达水平.结果 Gal - PEG - PEI组对HLA -E基因的沉默效率为55%,显著高于PEG - PEI组、Lipofectamine2000组、裸psiRNA组和阴性对照组(P<0.01).Gal-PEG-PEI组的沉默作用可持续7d.结论 经一步法Real - time RT - PCR检测,Gal - PEG - PEI/psiRNA纳米粒对HepG2细胞HLA-E基因有较高的沉默效率.
更多Objective To evaluate interference efficiency of HLA - E mRNA expression in HepG2 cell transfected with different psiRNA carrier using one - step real time RT - PCR method.Methods HepG2 cells were transfected by Gal - PEG - PEI/psiRNA,Gal - PEG - PEI/NC - psiRNA,PEG- PEI/psiRNA,Lipofectamine 2000/psiRNA and naked -psiRNA respectively.Total mRNA of HepG2 cells was extracted.The expression levels of HLA - E mRNA were detected by one - step real time RT - PCR.Results The interference efficiency of psiRNA mediated by Gal - PEG- PEI targetting HLA - E was 55% on mRNA expression level.It was distinctively higher than those of Lipofectamine 2000,PEG -PEI and naked -psiRNA ( P < 0.01 ),and its interference effect can last for seven days.Conclusions The Gal -PEG- PEI/psiRNA nanospheres display perfect hepatocyte -targeting ability and have higher interference efficiency to HLA- E gene in HepG2 cells detected by one -step real time RT- PCR.
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