%0 Journal Article
%T Influence of RT-qPCR primer position on EGFR interference efficacy in lung cancer cells
%A Gang Chen
%A Peter Kronenberger
%A Erik Teugels
%A Jacques De Gr¨¨ve
%J Biological Procedures Online
%D 2010
%I BioMed Central
%R 10.1186/1480-9222-13-1
%X 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.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 siRNA recognition sequence.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 (RT-qPCR) 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 level. A direct measurement at the mRNA level is therefore often the preferred method to more directly verify that RNAi is effectively decreasing the amount of the transcript. Real-time RT-qPCR is the "gold" standard for measuring steady-state mRNA levels. Hence, an accurate measurement method of the mRNA knockdown is needed. There are indications that mRNAs are not completely degraded after 24 hrs of RNAi exposure [2]. Therefore the location of the primer might be relevant as some primer sets may amplify remaining cleavage products, leading to an underestimation of the RNAi efficacy [3]. Despite this, numerous publications on RNAi with RT-qPCR do not evaluate the primers choice. The lack of precise criteria for choosing the target sequence for RT-qPCR amplification is surprising. Here, evidence is
%U http://www.biologicalproceduresonline.com/content/13/1/1