Parallel monitoring of RNA abundance, localization and compactness with correlative single molecule FISH on LR White embedded samples

Please always quote using this URN: urn:nbn:de:bvb:20-opus-230647
  • Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin.Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans.show moreshow less

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Metadaten
Author: Susanne KramerORCiD, Elisabeth Meyer-Natus, Christian Stigloher, Hanna Thoma, Achim SchnauferORCiD, Markus Engstler
URN:urn:nbn:de:bvb:20-opus-230647
Document Type:Journal article
Faculties:Fakultät für Biologie / Theodor-Boveri-Institut für Biowissenschaften
Language:English
Parent Title (English):Nucleic Acids Research
Year of Completion:2021
Volume:49
Issue:3
Article Number:e14
Source:Nucleic Acids Research, 2021, 49(3):e14. doi: 10.1093/nar/gkaa1142
DOI:https://doi.org/10.1093/nar/gkaa1142
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Tag:mRNA
Release Date:2021/04/21
Collections:Open-Access-Publikationsfonds / Förderzeitraum 2020
Licence (German):License LogoCC BY-NC: Creative-Commons-Lizenz: Namensnennung, Nicht kommerziell 4.0 International