Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immunostaining.
RNA
; 28(3): 433-446, 2022 03.
Article
en En
| MEDLINE
| ID: mdl-34949721
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 mRNA and allowing visualization of levels that escape detection using conventional procedures. The optimized protocol proved effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids in cultured cells.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
ARN
/
Técnica del Anticuerpo Fluorescente
/
Hibridación Fluorescente in Situ
Tipo de estudio:
Guideline
Límite:
Humans
Idioma:
En
Revista:
RNA
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2022
Tipo del documento:
Article
País de afiliación:
Alemania