Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immuno-staining.
- Prasath Paramasivam1,
- Martin Stoter2,
- Eloina Corradi3,
- Irene Dalla Costa3,
- Andreas Hoijer4,
- Stefano Bartesaghi5,
- Alan Sabirsh4,
- Lennart Lindfors4,
- Marianna Yanez Arteta6,
- Peter Nordberg7,
- Shalini Andersson8,
- Marie-Laure Baudet3,
- Marc Bickle9 and
- Marino Zerial9,10
- 1 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307, Dresden, Germany;
- 2 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108 01307. Dresden, Germany.;
- 3 Department CIBIO, University of Trento, Via Sommarive 9 Povo, Trento, Italy, 38123;
- 4 Advanced Drug Delivery, Pharmaceutical Science R&D, AstraZeneca, Gothenburg, Sweden;
- 5 Bioscience Metabolism, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden;
- 6 Advanced Drug Delivery, Pharmaceutical Science R&D, AstraZeneca, Gothenburg, Sweden.;
- 7 Medicinal Chemistry, Research and Early Development, Cardiovascular, Renal and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.;
- 8 Oligonucleotide Discovery, Discovery Sciences R&D, AstraZeneca, Gothenburg, Sweden.;
- 9 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307, Dresden, Germany.
- ↵* Corresponding author; email: zerial{at}mpi-cbg.de
Abstract
Detection of nucleic acids within sub-cellular 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 immuno-staining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels which 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.
Keywords
- Received July 6, 2021.
- Accepted December 12, 2021.
- Published by Cold Spring Harbor Laboratory Press for the RNA Society
This article, published in RNA, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
