Liu Wei-li, Stevenson Mark, Seymour Leonard W, Fisher Kerry D
Department of Clinical Pharmacology, University of Oxford, Headington, Oxford, UK.
Nucleic Acids Res. 2009 Jan;37(1):e4. doi: 10.1093/nar/gkn903. Epub 2008 Nov 12.
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.
我们基于小干扰RNA（siRNA）与同源DNA引物竞争退火至模板DNA，开发了一种基于聚合酶链反应（PCR）的siRNA定量方法，无需事先将RNA转化为互补DNA（cDNA）。引物和探针设计用于扩增人乳头瘤病毒E6或增强型绿色荧光蛋白基因区域。在确认siRNA不能作为扩增子生成的引物后，确定了模板DNA量（0.1 - 50 ng）与阈值循环数（Ct）之间呈线性关系的最低竞争引物浓度（6.25 nM）。在此条件下，向竞争性定量PCR（cqPCR）中加入序列特异性siRNA，导致Ct值呈剂量依赖性线性增加。与易受核酸内切酶影响的未修饰siRNA不同，2'-O-甲基核糖修饰的siRNA在血清中仍保留抑制模板扩增的能力。带有错配或截短的siRNA无法抑制模板扩增，证实了序列特异性以及区分降解和未降解siRNA序列的能力。使用脂质体转染试剂或His6可还原聚合物将E6 siRNA递送至C33 - A细胞后，通过cqPCR对siRNA摄取进行定量，结果显示摄取呈剂量依赖性。我们预计，cqPCR将能够准确测定体内递送后siRNA的药代动力学，极大地促进治疗性siRNA递送策略的开发。
