分子生物实验作业.pptVIP

Influence of RT-qPCR primer position on EGFR interference efficacy in lung cancer cells主题-肺癌细胞中在表皮生长因子受体干扰效能上RT-qPCR引物位置的影响。 Abstract Background: Real-time quantitative RT-PCR (RT-qPCR) is a “gold” standard for measuring steady state mRNA levels in RNA interference assays. The knockdown of the epidermal growth factor receptor (EGFR) gene with eight individual EGFR small interfering RNAs (siRNAs) was estimated by RT-qPCR using three different RT-qPCR primer sets. Results: Our results indicate that accurate measurement of siRNA efficacy by RT-qPCR requires careful attention for the selection of the primers used to amplify the target EGFR mRNA. Conclusions: We conclude that when assessing siRNA efficacy with RT-qPCR, more than one primer set targeting different regions of the mRNA should be evaluated and at least one of these primer sets should amplify a region encompassing the siRN recognition sequence. 摘要翻译 背景：实时定量逆转录聚合酶链反应技术(RT-qPCR)是在RNA干扰实验中测量稳态mRNA水平的“黄金”标准。表皮生长因子受体(EGFR)基因的敲除和八个特殊(EGFR)的小干扰RNA (siRNA)通过RT-qPCR来估测，这个RT-qPCR是三个不同的RT-qPCR引物集合。 结果：我们的研究结果表明：通过RT-qPCR的siRNA效能的精确测量要求密切注意引物的选择，引物是用来扩大EGFR mRNA.目标。 结论：我们得出的结论是当用RT-qPCR评估siRNA效能时，mRNA的不同区域上不只一个引物设置目标应当被估测并且这些引物集合中至少一个应当扩大一个区域包含siRNA识别序列。 Background RNA interference (RNAi) can mediate a short-term or prolonged silencing of gene expression at the RNA and protein level. Knockdown efficiency is typically measured at the mRNA level by quantitative RT-PCR (RTqPCR) or estimated at the protein level by immunoblot, enzyme-linked immunosorbent assay (ELISA) or immunohistochemistry (IHC) [1]. Many prefer measuring the relevant protein with immunoblot directly, because protein knockdown is most relevant to the observable phenotype under study. However, in practice, a suitable antibody to a given target protein may not always be readily available or will not allow a quantitative estimate of the magnitude of the effect of RNAi. The long turnover time of many proteins may underestimate the RNAi effect at the mRNA lev