Cargando…
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...
Autores principales: | , , , , |
---|---|
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 |
_version_ | 1782206946485469184 |
---|---|
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. |
format | Online Article Text |
id | pubmed-3123217 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT herbertmike theimportanceofrtqpcrprimerdesignforthedetectionofsirnamediatedmrnasilencing AT coppietersnatacha theimportanceofrtqpcrprimerdesignforthedetectionofsirnamediatedmrnasilencing AT lashamannette theimportanceofrtqpcrprimerdesignforthedetectionofsirnamediatedmrnasilencing AT caohelen theimportanceofrtqpcrprimerdesignforthedetectionofsirnamediatedmrnasilencing AT reidglen theimportanceofrtqpcrprimerdesignforthedetectionofsirnamediatedmrnasilencing AT herbertmike importanceofrtqpcrprimerdesignforthedetectionofsirnamediatedmrnasilencing AT coppietersnatacha importanceofrtqpcrprimerdesignforthedetectionofsirnamediatedmrnasilencing AT lashamannette importanceofrtqpcrprimerdesignforthedetectionofsirnamediatedmrnasilencing AT caohelen importanceofrtqpcrprimerdesignforthedetectionofsirnamediatedmrnasilencing AT reidglen importanceofrtqpcrprimerdesignforthedetectionofsirnamediatedmrnasilencing |