Kinetic analysis of the intracellular processing of siRNAs by confocal microscopy

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 siRNA...

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Bibliographic Details
Published inMicroscopy Vol. 69; no. 6; p. 401
Main Authors Vocelle, Daniel, Chesniak, Olivia M, Smith, Milton R, Chan, Christina, Walton, S Patrick
Format Journal Article
LanguageEnglish
Published England 03.12.2020
Subjects
HeLa
intracellular trafficking
siRNA
kinetics
confocal microscopy
delivery vehicles
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Summary: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.
ISSN:2050-5701
DOI:10.1093/jmicro/dfaa031