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

Susanne Kramer, Elisabeth Meyer-Natus, Christian Stigloher, Hanna Thoma, Achim Schnaufer, Markus Engstler

Research output: Contribution to journalArticlepeer-review

Abstract

Single mRNA molecules are frequently detected by single molecule fluorescence insitu hybridisation (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 hybridised 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 localisation 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.
Original languageEnglish
Article numbergkaa1142
JournalNucleic Acids Research
DOIs
Publication statusPublished - 4 Dec 2020

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