摘要
目的评价实时荧光定量逆转录聚合酶链反应(RT-PCR)法检测小干扰RNA(siRNA)表达载体抑制人肾癌786-0细胞G250 mRNA表达的可靠性,以建立稳定的RNAi技术。方法体外合成四条针对G250基因的siRNA,并设计阴性对照和空白对照,转染肾癌786-0细胞株;根据G250 mRNA的序列设计特异性引物,荧光染料SYBR Green Ⅰ法定量RT-PCR测定G250 mRNA的表达情况。结果四条siRNA分别使G250 mRNA表达量降低了35.56%、49.27%、45.88%、65.13%,阴性对照和空白对照组无明显变化。结论实时荧光定量RT-PCR技术可以准确地检测mRNA的表达,可以用来评价siRNA的有效性。
Objective To evaluate the reliability of real-time fluorescence quantitative RT-PCR in detecting G250 mRNA expression in G250 gene down-knocked renal cell carcinoma cell line 786-0 by RNA interference（RNAi） technique,in order to establish stable RNAi.Methods Four siRNAs against the G250 gene,negative control and blank were designed and transfected into 786-0 cells respectively,The specific primers were designed according to the sequence of G250 mRNA,and the fluorescence dye SYBR Green I were used for RT-PCR amplificatiom,then amount of G250 mRNA in the 786-0 cells after transfection was quantitated.Results 35.56%、49.27%、45.88%、65.13% silencing of G250 mRNA expression were observed after transfecting with four siRNA respectively,but negative control and blank were not decrease obviously.Conclusion These results demonstrated that real-time fluorescence quantitative RT-PCR technology could detect the mRNA expression accurately,it can be used in the evaluating effect of RNAi.
出处
《中国实验诊断学》
北大核心
2011年第3期404-406,共3页
Chinese Journal of Laboratory Diagnosis
