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一种高度敏感的干环RT-qPCR方法,用于研究siRNA细胞内药理动力学和药理动力学

Lin Chen¹, Caroline Bosmajian¹, Sukyung Woo¹

¹Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, NY 14214, United States.

Biology methods & protocols

|May 24, 2024

概括

一种新的干环RT-qPCR方法准确地量化了细胞中的细胞内小干扰RNA (siRNA). 这种经过验证的技术可以精确测量siRNA的药理动力学和药理动力学,用于治疗开发.

科学领域:

生物化学生化学
分子生物学分子生物学
药理学药理学是指药理学的学科.

背景情况:

小干扰RNA (siRNA) 在疾病治疗中对基因沉默至关重要.
了解siRNA的细胞内行为 (药理动力学/药理动力学) 对治疗开发至关重要.
需要准确的量化方法来研究siRNA细胞内动力学.

研究的目的:

建立和验证用于细胞内siRNA量化的干环定量PCR方法.
评估影响siRNA测量精度的因素.
为了使siRNA PK/PD属性的详细表征.

主要方法:

开发并验证了一种干环逆转录定量聚合酶链反应 (RT-qPCR) 试验.
直接细胞溶解与基于固定化的提取用于siRNA恢复的比较.
优化了RT原料设计和预回暖步骤,以提高灵敏度.
利用miR-16和蛋白质度用于细胞质和RISC载荷siRNA的正常化.

主要成果:

使用干环RT-qPCR的直接细胞溶解显示了siRNA量化方面的优异可重复性.
已确定的量化范围:0.000220 fmole (未修改的siRNA) 和0.0220 fmole (修改的siRNA).
在样本处理和储存过程中发现了siRNA损失的显著变化.

结论:

开发的干环RT-qPCR方法为细胞内siRNA量化提供了可靠的工具.
这种方法促进了siRNA PK/PD的研究,这对于推进siRNA疗法至关重要.
优化的协议和规范化策略提高了siRNA测量的准确性和可重复性.

关键词:

在RISC中加载RISC.细胞内部的药理动力学 siRNA 是一个RNA.树干循环RT-qPCR是什么意思

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相关概念视频

siRNA - Small Interfering RNAs

siRNA - Small Interfering RNAs

Small interfering RNAs, or siRNAs, are short regulatory RNA molecules that can silence genes post-transcriptionally, as well as the transcriptional level in some cases. siRNAs are important for protecting cells against viral infections and silencing transposable genetic elements.
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the...