The goal of this study was to develop a reverse transcription quantitative polymerase chain reaction (RT-qPCR) method for the accurate quantification of chemically modified small interfering RNA (siRNA) including but not restricted to thermally destabilizing modifications such as glycol nucleic acid (GNA). RT-qPCR was found to be superior to mass spectrometry-based siRNA detection in terms of sensitivity and throughput. However, mass spectrometry is still the preferred method when specific metabolite detection is required and is also insensitive to siRNA chemical modifications such as GNA. The RT-qPCR approach can be optimized to take chemical modifications into account and works robustly in different matrices without optimization, unlike mass spectrometry. RT-qPCR and mass spectrometry both have their strengths and weaknesses for the detection of siRNA and must be used appropriately depending on the questions at hand. Considerations such as desired throughput, assay sensitivity, and metabolite identification must be weighed when choosing which methodology to apply.

中文翻译：

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支持药代动力学和药物作用机制的 RT-qPCR 方法，以促进 RNAi 疗法的发展。

本研究的目标是开发一种逆转录定量聚合酶链反应 (RT-qPCR) 方法，用于准确定量化学修饰的小干扰 RNA (siRNA)，包括但不限于热不稳定修饰，如乙二醇核酸 (GNA) . 发现 RT-qPCR 在灵敏度和通量方面优于基于质谱的 siRNA 检测。然而，当需要特定代谢物检测并且对 siRNA 化学修饰（如 GNA）不敏感时，质谱仍然是首选方法。与质谱不同，RT-qPCR 方法可以优化以考虑化学修饰，并且无需优化即可在不同基质中稳健运行。RT-qPCR 和质谱法在检测 siRNA 方面各有优缺点，必须根据手头的问题适当使用。在选择应用哪种方法时，必须权衡所需的通量、测定灵敏度和代谢物鉴定等考虑因素。