Abstract
RNA interference (RNAi) is a powerful tool for studying functions of candidate genes in both model and nonmodel organisms and a promising technique for therapeutic applications. Successful application of this technique relies on the accuracy and reliability of methods used to quantify gene knockdown. With the limitation in the availability of antibodies for detecting proteins, quantitative PCR (qPCR) remains the preferred method for quantifying target gene knockdown after dsRNA treatment. We evaluated how qPCR primer binding site and target gene expression levels affect quantification of intact mRNA transcripts following dsRNA‐mediated RNAi. The use of primer pairs targeting the mRNA sequence within the dsRNA target region failed to reveal a significant decrease in target mRNA transcripts for genes with low expression levels, but not for a highly expressed gene. By contrast, significant knockdown was detected in all cases with primer pairs targeting the mRNA sequence extending beyond the dsRNA target region, regardless of the expression levels of the target gene. Our results suggest that at least for genes with low expression levels, quantifying the efficiency of dsRNA‐mediated RNAi with primers amplifying sequences completely contained in the dsRNA target region should be avoided due to the risk of false‐negative results. Instead, primer pairs extending beyond the dsRNA target region of the mRNA transcript sequences should be used for accurate and reliable quantification of silencing efficiency.
Highlights
The discovery of RNA interference (RNAi) revolutionized the study of gene functions in eukaryotes
Our results suggest that at least for genes with low expression levels, quan‐ tifying the efficiency of double‐stranded RNA (dsRNA)‐mediated RNAi with primers amplifying sequences completely contained in the dsRNA target region should be avoided due to the risk of false‐negative results
To confirm that the observed phenotypic changes are due to the knockdown of tar‐ get genes and not off‐target effects, the concentrations of residual target gene transcripts and proteins are measured with RT‐quantitative PCR (qPCR) and western blotting, respectively
Summary
The discovery of RNA interference (RNAi) revolutionized the study of gene functions in eukaryotes. RNAi regulates development and physiology, sup‐ presses transposon activity, and provides defense against RNA virus infections in many organisms using endogenously expressed microR‐ NAs or exogenously introduced viral dsRNA (Ambros, 2004; Obbard, Gordon, Buck, & Jiggins, 2009) This mechanism can be exploited artificially to study functions of endogenous eukaryotic genes of interest through the introduction of synthetic dsRNA or siRNA molecules that trigger the host's natural RNAi machinery to silence the respective genes, which allows investigation into their specific functions. We report that the use of qPCR primers targeting a sequence that is completely contained within the dsRNA construct can lead to false negatives or an under‐ estimation of gene knockdown in genes with low expression levels
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