A highly sensitive stem-loop RT-qPCR method to study siRNA intracellular pharmacokinetics and pharmacodynamics

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS
Lin Chen, Caroline Bosmajian, Sukyung Woo
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引用次数: 0

Abstract

Small interfering RNA (siRNA) is a powerful tool for sequence-specific silencing of disease-related genes. In this study, we established and validated a stem-loop reverse transcription real-time polymerase chain reaction (RT-qPCR) method applicable for both chemically unmodified and modified siRNA, aiming to elucidate mechanistic intracellular pharmacokinetic and pharmacodynamic (PK/PD) properties of siRNA. We conducted a comprehensive evaluation of factors affecting intracellular siRNA quantification. Our study revealed that immobilization-based siRNA extraction introduced high variation, making it unsuitable for absolute quantification. Conversely, direct cell lysis followed by stem-loop RT-qPCR demonstrated excellent reproducibility, with a quantification range from 0.0002 to 20 femtomole (fmole) for unmodified siRNA and 0.02 to 20 fmole for modified siRNA. The design of a 6-basepair overlapping RT primer facilitated the distinction of full-length antisense from its 3’ metabolites, and pre-annealing of antisense to RT primer enhanced sensitivity and reproducibility. Differences in siRNA loss during storage and sample processing were noted among microcentrifuge tubes from various manufacturers. Endogenous miR-16 served as a reference for normalizing cytoplasmic siRNA, while protein concentration post-immunoprecipitation lysis was used to normalize RISC-loaded siRNA levels. This method successfully enabled a detailed characterization of the time profiles of cytoplasmic and RISC-loaded siRNA, advancing of the in vitro-in vivo translation of siRNA therapeutics.
研究 siRNA 细胞内药代动力学和药效学的高灵敏度茎环 RT-qPCR 方法
小干扰 RNA(siRNA)是序列特异性沉默疾病相关基因的有力工具。在这项研究中,我们建立并验证了一种适用于化学未修饰和修饰 siRNA 的干环反向转录实时聚合酶链反应(RT-qPCR)方法,旨在阐明 siRNA 的细胞内药代动力学和药效学(PK/PD)机理特性。我们对影响细胞内 siRNA 定量的因素进行了全面评估。我们的研究发现,基于固定化技术的 siRNA 提取会带来很大的变化,因此不适合绝对定量。相反,直接裂解细胞后进行干环 RT-qPCR 则表现出极佳的重现性,未修饰 siRNA 的定量范围为 0.0002 至 20 飞摩尔(fmole),修饰 siRNA 的定量范围为 0.02 至 20 飞摩尔。6 碱基对重叠 RT 引物的设计有助于区分全长反义和其 3' 代谢产物,反义与 RT 引物的预退火提高了灵敏度和重现性。不同厂家生产的微离心管在储存和样品处理过程中 siRNA 的损失存在差异。内源性 miR-16 可作为细胞质 siRNA 正常化的参考,而免疫沉淀裂解后的蛋白质浓度可用于 RISC 加载的 siRNA 水平的正常化。这种方法成功地详细描述了细胞质和 RISC 负载 siRNA 的时间曲线,推进了 siRNA 疗法的体外-体内转化。
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
ACS Applied Bio Materials
ACS Applied Bio Materials Chemistry-Chemistry (all)
CiteScore
9.40
自引率
2.10%
发文量
464
期刊介绍: ACS Applied Bio Materials is an interdisciplinary journal publishing original research covering all aspects of biomaterials and biointerfaces including and beyond the traditional biosensing, biomedical and therapeutic applications. The journal is devoted to reports of new and original experimental and theoretical research of an applied nature that integrates knowledge in the areas of materials, engineering, physics, bioscience, and chemistry into important bio applications. The journal is specifically interested in work that addresses the relationship between structure and function and assesses the stability and degradation of materials under relevant environmental and biological conditions.
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