title: Quantification of siRNA using competitive qPCR.
abstract: 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.
authors.affiliation: Department of Clinical Pharmacology, University of Oxford, Headington, Oxford, UK.
authors.forename: Wei-li
authors.initials: WL
authors.lastname: Liu
authors.forename: Mark
authors.initials: M
authors.lastname: Stevenson
authors.forename: Leonard W
authors.initials: LW
authors.lastname: Seymour
authors.forename: Kerry D
authors.initials: KD
authors.lastname: Fisher
mesh_terms: D045744:Cell Line, Tumor; D017931:DNA Primers; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D034741:RNA, Small Interfering / Q000032:analysis*; D012097:Repressor Proteins / Q000235:genetics; D014162:Transfection
chemical_list: D017931:DNA Primers; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D034741:RNA, Small Interfering; D012097:Repressor Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins