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Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry

A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplificati...

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Autores principales: Channathodiyil, Prasanna, Houseley, Jonathan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822327/
https://www.ncbi.nlm.nih.gov/pubmed/33481798
http://dx.doi.org/10.1371/journal.pone.0240769
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author Channathodiyil, Prasanna
Houseley, Jonathan
author_facet Channathodiyil, Prasanna
Houseley, Jonathan
author_sort Channathodiyil, Prasanna
collection PubMed
description A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal—a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells.
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spelling pubmed-78223272021-01-29 Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry Channathodiyil, Prasanna Houseley, Jonathan PLoS One Research Article A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal—a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells. Public Library of Science 2021-01-22 /pmc/articles/PMC7822327/ /pubmed/33481798 http://dx.doi.org/10.1371/journal.pone.0240769 Text en © 2021 Channathodiyil, Houseley http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Channathodiyil, Prasanna
Houseley, Jonathan
Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry
title Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry
title_full Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry
title_fullStr Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry
title_full_unstemmed Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry
title_short Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry
title_sort glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822327/
https://www.ncbi.nlm.nih.gov/pubmed/33481798
http://dx.doi.org/10.1371/journal.pone.0240769
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