Autoren:	Onchuru, Thomas Kaltenpoth, Martin
Titel:	Quantitative PCR primer design affects quantification of dsRNA-mediated gene knockdown
Online-Publikationsdatum:	16-Dez-2019
Erscheinungsdatum:	2019
Sprache des Dokuments:	Englisch
Zusammenfassung/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-Sachgruppe:	570 Biowissenschaften 570 Life sciences
Veröffentlichende Institution:	Johannes Gutenberg-Universität Mainz
Organisationseinheit:	FB 10 Biologie
Veröffentlichungsort:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-243
URN:	urn:nbn:de:hebis:77-publ-594768
Version:	Published version
Publikationstyp:	Zeitschriftenaufsatz
Nutzungsrechte:	CC BY
Informationen zu den Nutzungsrechten:	hhttps://creativecommons.org/licenses/by/4.0/
Zeitschrift:	Ecology and evolution 9 14
Seitenzahl oder Artikelnummer:	8187 8192
Verlag:	John Wiley & Sons, Inc.
Verlagsort:	S.l.
Erscheinungsdatum:	2019
ISSN:	2045-7758
URL der Originalveröffentlichung:	http://dx.doi.org/10.1002/ece3.5387
DOI der Originalveröffentlichung:	10.1002/ece3.5387
Enthalten in den Sammlungen:	JGU-Publikationen