ArticleBMC research notes2017
Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines.
Article in BMC research notes, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed.
- Real-time, cross-modal genotype mapping of free-movingScience advances · 2026Article
- An RNA interference approach for functional studies in the sea urchin and its use in analysis of nodal signaling gradients.Developmental biology · 2024Article
- An RNA interference approach for functional studies in the sea urchin and its use in analysis of Nodal signaling gradients.bioRxiv : the preprint server for biology · 2024Article
- Intrasexual cuticular hydrocarbon dimorphism in a wasp sheds light on hydrocarbon biosynthesis genes in Hymenoptera.Communications biology · 2023Article
- Combined p53- and PTEN-deficiency activates expression of mesenchyme homeobox 1 (MEOX1) required for growth of triple-negative breast cancer.The Journal of biological chemistry · 2020Article
- Quantitative PCR primer design affects quantification of dsRNA-mediated gene knockdown.Ecology and evolution · 2019Article
- Individual components of the SWI/SNF chromatin remodelling complex have distinct roles in memory neurons of theDisease models & mechanisms · 2019Article
- The histone methyltransferase G9a regulates tolerance to oxidative stress-induced energy consumption.PLoS biology · 2019Article
- Vitellogenin-like A-associated shifts in social cue responsiveness regulate behavioral task specialization in an ant.PLoS biology · 2018Article
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4 authors.
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Abstract
objectiveRNA interference is employed extensively in Drosophila research to study gene function within a specific cell-type or tissue. Thousands of transgenic Drosophila lines have been generated to express double stranded RNA for gene knockdown; however, no standardized method exists for quantifying their knockdown efficiency. Since antibodies are not available for many proteins, quantitative real-time PCR is often used. Here, we explore how primer design and RNA isolation method can influence detection of gene knockdown using qPCR.
resultsWe tested differences in detected gene knockdown efficiency when using purified polyadenylated mRNA or total RNA as templates for cDNA synthesis. We also tested two different primer locations for each gene: one to amplify a region 5' of the RNAi cut site, and one to amplify a region 3' of the cut site. Consistently, the strongest gene knockdown was detected when qPCR was performed using 5' primer sets in combination with mRNA-derived cDNA. Our results indicate that detection of undegraded mRNA cleavage fragments can result in underestimation of true knockdown efficiency for a RNAi construct. Purification of polyadenylated mRNA, combined with primers designed to amplify the non-polyadenylated 5' mRNA cleavage fragment can avoid this problem.
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