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Nucleic Acids Research Advance Access originally published online on November 12, 2008
Nucleic Acids Research 2009 37(1):e4; doi:10.1093/nar/gkn903
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Nucleic Acids Research, 2009, Vol. 37, No. 1 e4
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Quantification of siRNA using competitive qPCR

Wei-li Liu, Mark Stevenson*, Leonard W. Seymour and Kerry D. Fisher

Department of Clinical Pharmacology, Old Road Campus Research Building, University of Oxford, Old Road Campus, off Roosevelt Drive, Headington, Oxford OX3 7DQ, UK

*To whom correspondence should be addressed. Tel: +44 1865 617041; Fax: +44 1865 617028; Email: mark.stevenson{at}clinpharm.ox.ac.uk

Received March 10, 2008. Revised October 27, 2008. Accepted October 28, 2008.

We have developed a PCR-based short interfering RNA (siRNA) quantification method based on competition between siRNA and a homologous DNA primer for annealing to template DNA, avoiding the requirement for prior conversion of RNA to cDNA. Primers and probe were designed to amplify regions of the human papillomavirus E6 or enhanced green fluorescent protein genes. Having confirmed siRNA could not act as primer for amplicon generation, the lowest competing primer concentration yielding a linear relationship between template DNA amount (0.1–50 ng) and cycle of threshold (Ct) was determined (6.25 nM). Under these conditions addition of sequence-specific siRNA to the competitive quantitative PCR (cqPCR), resulted in a dose-dependent linear increase in Ct value. 2'-O-methyl ribose-modified siRNA retained an ability to inhibit template amplification in serum, unlike unmodified siRNAs that were susceptible to endonucleases. Mismatch-bearing or truncated siRNAs failed to inhibit template amplification confirming sequence specificity and an ability to discriminate between degraded and non-degraded siRNA sequences. Following delivery of E6 siRNA to C33-A cells using oligofectamine or His6 reducible polymers, siRNA uptake was quantified by cqPCR, revealing dose-dependent uptake. We anticipate that cqPCR will allow accurate determination of siRNA pharmacokinetics following in vivo delivery, greatly facilitating development of therapeutic siRNA delivery strategies.


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