Kinetic analysis of the intracellular processing of siRNAs by confocal microscopy
Kinetic analysis of the intracellular processing of siRNAs by confocal microscopy
复制标题
通过共聚焦显微镜对 siRNA 细胞内加工进行动力学分析
DOI：
10.1093/jmicro/dfaa031
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发表时间：
2020
期刊：
影响因子：
1.8
通讯作者：
Walton, S Patrick
中科院分区：
文献类型：
--
作者：
Vocelle, Daniel;Chesniak, Olivia M;Smith, Milton R;Chan, Christina;Walton, S Patrick
在这里，我们描述了一种使用自动显微镜控制和图像采集来跟踪含有小干扰RNA （siRNA）复合物的细胞内处理的方法，以最大限度地减少用户的工作量和时间。该技术利用荧光共定位技术监测二氧化硅纳米颗粒在细胞内不同位置递送的双标记荧光sirna，包括早/晚核内体、快/慢循环核内体、溶酶体和内质网。结合siRNA与每个细胞内位置的时间关联，我们重建了siRNA加工中使用的细胞内途径，并展示了这些途径如何根据递送载体的化学成分而变化。
Here, we describe a method for tracking intracellular processing of small interfering RNA (siRNA) containing complexes using automated microscopy controls and image acquisition to minimize user effort and time. This technique uses fluorescence colocalization to monitor dual-labeled fluorescent siRNAs delivered by silica nanoparticles in different intracellular locations, including the early/late endosomes, fast/slow recycling endosomes, lysosomes and the endoplasmic reticulum. Combining the temporal association of siRNAs with each intracellular location, we reconstructed the intracellular pathways used in siRNA processing, and demonstrate how these pathways vary based on the chemical composition of the delivery vehicle.