Supplementary data: Utilizing droplet digital polymerase chainreaction for siRNA quantitation in rodentplasma and tissue via stem-loop reversetranscription

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.

背景：小干扰核糖核酸（siRNA）是一种极具前景的治疗方式，自2018年以来已获得美国食品药品监督管理局（FDA）多项批准，另有更多寡核苷酸资产处于临床开发阶段。为支持siRNA的发现与开发，需建立稳健且灵敏的生物分析定量平台，以评估药代动力学/药效动力学（pharmacokinetic/pharmacodynamic）关系及毒理学特征。与其他分析平台相比，微滴式数字聚合酶链式反应（Droplet digital PCR）具有更高的灵敏度和通量，且对基质效应的敏感性更低。
方法：作者依据生物分析方法验证指南，开发了一种茎环逆转录（stem-loop reverse transcription）微滴式数字聚合酶链式反应方法，用于检测小鼠血浆及肝提取物中的siRNA。
结论：该新开发的检测方法已被证明是其他平台的优良替代方案，具有更高的灵敏度优势，其动态范围为390至400,000拷贝/反应，且可用于支持美国食品药品监督管理局（FDA）的研究性新药申报相关应用。