Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immunostaining.

ABSTRACT: Detection of nucleic acids within subcellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is particularly important for the delivery of nucleic acid therapeutics, which depends on endocytic uptake and endosomal escape. However, the current protocols fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization, and immunostaining of a number of organelle markers, achieving quantitative retention of mRN

SUBMITTER: Paramasivam P 

PROVIDER: S-EPMC8848937 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature