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Authors: Onchuru, Thomas
Kaltenpoth, Martin
Title: Quantitative PCR primer design affects quantification of dsRNA-mediated gene knockdown
Online publication date: 16-Dec-2019
Year of first publication: 2019
Language: english
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.
DDC: 570 Biowissenschaften
570 Life sciences
Institution: Johannes Gutenberg-Universität Mainz
Department: FB 10 Biologie
Place: Mainz
URN: urn:nbn:de:hebis:77-publ-594768
Version: Published version
Publication type: Zeitschriftenaufsatz
License: CC BY
Information on rights of use: h
Journal: Ecology and evolution
Pages or article number: 8187
Publisher: John Wiley & Sons, Inc.
Publisher place: S.l.
Issue date: 2019
ISSN: 2045-7758
Publisher URL:
Publisher DOI: 10.1002/ece3.5387
Appears in collections:JGU-Publikationen

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