Utilizing droplet digital polymerase chain reaction for siRNA quantitation in rodent plasma and tissue via stem-loop reverse transcription.

IF 1.9 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Bioanalysis Pub Date : 2024-03-01 Epub Date: 2024-02-21 DOI:10.4155/bio-2023-0228

Megan K Turski, Matthew E Albertolle

引用次数: 0

Abstract

Background: siRNA is a promising therapeutic modality highlighted by several US FDA approvals since 2018, with many more oligonucleotide assets in clinical development. To support siRNA discovery and development, robust and sensitive quantitative platforms for bioanalysis must be established to assess pharmacokinetic/pharmacodynamic relationships and toxicology. Droplet digital PCR offers improved sensitivity and throughput, as well as reduced susceptibility to matrix effects, compared with other analytical platforms. Methodology: The authors developed a stem-loop reverse transcription droplet digital PCR method to measure siRNA in mouse plasma and liver extract using bioanalytical method qualification guidelines. Conclusion: This newly developed assay has been demonstrated to be a superior alternative to other platforms, with the added benefit of greater sensitivity, with dynamic range from 390 to 400,000 copies/reaction and readiness for FDA investigational new drug-enabling applications.

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利用液滴数字聚合酶链式反应，通过干环逆转录对啮齿动物血浆和组织中的 siRNA 进行定量。

背景：siRNA 是一种前景广阔的治疗方式，自 2018 年以来，美国 FDA 批准了多项 siRNA，还有更多寡核苷酸资产正在进行临床开发。为支持 siRNA 的发现和开发，必须建立强大而灵敏的生物分析定量平台，以评估药代动力学/药效学关系和毒理学。与其他分析平台相比，液滴数字 PCR 提高了灵敏度和通量，并降低了对基质效应的敏感性。研究方法作者采用生物分析方法鉴定指南，开发了一种干环反转录液滴数字 PCR 方法，用于检测小鼠血浆和肝脏提取物中的 siRNA。结论这种新开发的检测方法已被证明是一种优于其他平台的替代方法，其优点是灵敏度更高，动态范围从 390 到 400,000 拷贝/反应，可用于美国食品及药物管理局的新药研发应用。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Bioanalysis BIOCHEMICAL RESEARCH METHODS-CHEMISTRY, ANALYTICAL

CiteScore

3.30

自引率

16.70%

发文量

审稿时长

2 months

期刊介绍： Reliable data obtained from selective, sensitive and reproducible analysis of xenobiotics and biotics in biological samples is a fundamental and crucial part of every successful drug development program. The same principles can also apply to many other areas of research such as forensic science, toxicology and sports doping testing. The bioanalytical field incorporates sophisticated techniques linking sample preparation and advanced separations with MS and NMR detection systems, automation and robotics. Standards set by regulatory bodies regarding method development and validation increasingly define the boundaries between speed and quality. Bioanalysis is a progressive discipline for which the future holds many exciting opportunities to further reduce sample volumes, analysis cost and environmental impact, as well as to improve sensitivity, specificity, accuracy, efficiency, assay throughput, data quality, data handling and processing. The journal Bioanalysis focuses on the techniques and methods used for the detection or quantitative study of analytes in human or animal biological samples. Bioanalysis encourages the submission of articles describing forward-looking applications, including biosensors, microfluidics, miniaturized analytical devices, and new hyphenated and multi-dimensional techniques. Bioanalysis delivers essential information in concise, at-a-glance article formats. Key advances in the field are reported and analyzed by international experts, providing an authoritative but accessible forum for the modern bioanalyst.