Examination of real-time polymerase chain reaction methods for the detection and quantification of modified siRNA
Examination of real-time polymerase chain reaction methods for the detection and quantification of modified siRNA
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DOI：
10.1016/j.ab.2008.05.001
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发表时间：
2008-08-01
影响因子：
2.9
通讯作者：
Beverly, Michael
中科院分区：
文献类型：
--
作者：
Stratford, Suzanne;Stec, Summer;Beverly, Michael
随着不断努力开发基于siRNA的治疗方法，需要高通量检测和定量siRNA。在这里，我们报告了四种基于逆转录酶RT-PCR的方法用于检测小鼠血浆和组织中2‘-脱氧-2’-氟和2‘-O-甲基修饰的治疗性siRNA。这些检测方法利用了聚合酶链式反应检测的动态范围、敏感性、特异性和高通量潜力。其中三种检测方法需要设计和优化siRNA特异的引物和/或探针，而第四种检测方法使用独立于siRNA序列的“通用”TaqMan探针，从而减少了方法开发时间和成本。对于通用方法，五种模型荧光素酶序列中的四种在小鼠血浆中的检测范围为500到5E(-5)pg/亩L。我们发现，通用RT-PCR方法具有与其他三种方法相当或更好的敏感性和特异性。通用设计提供了一种快速、灵敏和特异的分析方法，方法开发时间最短，非常适合于各种siRNA序列的高通量分析。(C)2008 Elsevier Inc.保留所有权利。
With the ongoing efforts to develop siRNA-based therapeutics, there is a need for high-throughput detection and quantification of siRNA. Here we report the application of four reverse-transcriptase RT-PCR-based assays for the detection of 2'-deoxy-2'-fluoro and 2'-O-methyl-modified therapeutic siRNA in mouse plasma and tissue. These assays take advantage of the dynamic range, sensitivity, specificity, and high-throughput potential found in PCR assays. Three of these assays require design and optimization of primers and/or probes specific to the siRNA while the fourth utilizes a "universal" TaqMan probe that is independent of the siRNA sequence, thereby reducing method development time and cost. For the universal assay the range of detection in mouse plasma was 500 to 5e(-5) pg/mu l for four of five model Luciferase sequences tested. We found that the universal RT-PCR assay had comparable or better sensitivity and specificity than the other three assays. The universal design provides a rapid, sensitive, and specific assay with minimal method development time that will be well suited for high-throughput analysis of various siRNA sequences. (C) 2008 Elsevier Inc. All rights reserved.