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The importance of RT-qPCR primer design for the detection of siRNA-mediated mRNA silencing

BACKGROUND: The use of RNAi to analyse gene function in vitro is now widely applied in biological research. However, several difficulties are associated with its use in vivo, mainly relating to inefficient delivery and non-specific effects of short RNA duplexes in animal models. The latter can lead...

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Autores principales: Herbert, Mike, Coppieters, Natacha, Lasham, Annette, Cao, Helen, Reid, Glen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3123217/
https://www.ncbi.nlm.nih.gov/pubmed/21612654
http://dx.doi.org/10.1186/1756-0500-4-148
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author Herbert, Mike
Coppieters, Natacha
Lasham, Annette
Cao, Helen
Reid, Glen
author_facet Herbert, Mike
Coppieters, Natacha
Lasham, Annette
Cao, Helen
Reid, Glen
author_sort Herbert, Mike
collection PubMed
description BACKGROUND: The use of RNAi to analyse gene function in vitro is now widely applied in biological research. However, several difficulties are associated with its use in vivo, mainly relating to inefficient delivery and non-specific effects of short RNA duplexes in animal models. The latter can lead to false positive results when real-time RT-qPCR alone is used to measure target mRNA knockdown. FINDINGS: We observed that detection of an apparent siRNA-mediated knockdown in vivo was dependent on the primers used for real-time RT-qPCR measurement of the target mRNA. Two siRNAs specific for RRM1 with equivalent activity in vitro were administered to A549 xenografts via intratumoural injection. In each case, apparent knockdown of RRM1 mRNA was observed only when the primer pair used in RT-qPCR flanked the siRNA cleavage site. This false-positive result was found to result from co-purified siRNA interfering with both reverse transcription and qPCR. CONCLUSIONS: Our data suggest that using primers flanking the siRNA-mediated cleavage site in RT-qPCR-based measurements of mRNA knockdown in vivo can lead to false positive results. This is particularly relevant where high concentrations of siRNA are introduced, particularly via intratumoural injection, as the siRNA may be co-purified with the RNA and interfere with downstream enzymatic steps. Based on these results, using primers flanking the siRNA target site should be avoided when measuring knockdown of target mRNA by real-time RT-qPCR.
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spelling pubmed-31232172011-06-25 The importance of RT-qPCR primer design for the detection of siRNA-mediated mRNA silencing Herbert, Mike Coppieters, Natacha Lasham, Annette Cao, Helen Reid, Glen BMC Res Notes Short Report BACKGROUND: The use of RNAi to analyse gene function in vitro is now widely applied in biological research. However, several difficulties are associated with its use in vivo, mainly relating to inefficient delivery and non-specific effects of short RNA duplexes in animal models. The latter can lead to false positive results when real-time RT-qPCR alone is used to measure target mRNA knockdown. FINDINGS: We observed that detection of an apparent siRNA-mediated knockdown in vivo was dependent on the primers used for real-time RT-qPCR measurement of the target mRNA. Two siRNAs specific for RRM1 with equivalent activity in vitro were administered to A549 xenografts via intratumoural injection. In each case, apparent knockdown of RRM1 mRNA was observed only when the primer pair used in RT-qPCR flanked the siRNA cleavage site. This false-positive result was found to result from co-purified siRNA interfering with both reverse transcription and qPCR. CONCLUSIONS: Our data suggest that using primers flanking the siRNA-mediated cleavage site in RT-qPCR-based measurements of mRNA knockdown in vivo can lead to false positive results. This is particularly relevant where high concentrations of siRNA are introduced, particularly via intratumoural injection, as the siRNA may be co-purified with the RNA and interfere with downstream enzymatic steps. Based on these results, using primers flanking the siRNA target site should be avoided when measuring knockdown of target mRNA by real-time RT-qPCR. BioMed Central 2011-05-25 /pmc/articles/PMC3123217/ /pubmed/21612654 http://dx.doi.org/10.1186/1756-0500-4-148 Text en Copyright ©2011 Reid et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Short Report
Herbert, Mike
Coppieters, Natacha
Lasham, Annette
Cao, Helen
Reid, Glen
The importance of RT-qPCR primer design for the detection of siRNA-mediated mRNA silencing
title The importance of RT-qPCR primer design for the detection of siRNA-mediated mRNA silencing
title_full The importance of RT-qPCR primer design for the detection of siRNA-mediated mRNA silencing
title_fullStr The importance of RT-qPCR primer design for the detection of siRNA-mediated mRNA silencing
title_full_unstemmed The importance of RT-qPCR primer design for the detection of siRNA-mediated mRNA silencing
title_short The importance of RT-qPCR primer design for the detection of siRNA-mediated mRNA silencing
title_sort importance of rt-qpcr primer design for the detection of sirna-mediated mrna silencing
topic Short Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3123217/
https://www.ncbi.nlm.nih.gov/pubmed/21612654
http://dx.doi.org/10.1186/1756-0500-4-148
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