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

Mike Herbert13, Natacha Coppieters14, Annette Lasham15, Helen Cao16 and Glen Reid127*

Author Affiliations

1 Genesis Research & Development Corporation, Ltd, PO Box 50, Auckland 1140, New Zealand

2 Department of Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand

3 Department of Structural Biology, School of Biological Sciences, University of Auckland, Auckland, New Zealand

4 Department of Pharmacology, University of Auckland, Auckland, New Zealand

5 Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand

6 CSL Limited, 45 Poplar Rd, Parkville VIC 3052, Australia

7 Asbestos Diseases Research Institute (ADRI), Bernie Banton Centre, University of Sydney, Hospital Road, Concord NSW 2139, Australia

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BMC Research Notes 2011, 4:148  doi:10.1186/1756-0500-4-148

Published: 25 May 2011



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.


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.


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.