Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

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

Prasanna Channathodiyil, View ORCID ProfileJonathan Houseley
doi: https://doi.org/10.1101/2020.10.05.326082
Prasanna Channathodiyil
Epigenetics Programme, Babraham Institute, Cambridge, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jonathan Houseley
Epigenetics Programme, Babraham Institute, Cambridge, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Jonathan Houseley
  • For correspondence: jon.houseley@babraham.ac.uk
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Preview PDF
Loading

Abstract

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.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
Back to top
PreviousNext
Posted October 05, 2020.
Download PDF
Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry
Prasanna Channathodiyil, Jonathan Houseley
bioRxiv 2020.10.05.326082; doi: https://doi.org/10.1101/2020.10.05.326082
Reddit logo Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools
Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry
Prasanna Channathodiyil, Jonathan Houseley
bioRxiv 2020.10.05.326082; doi: https://doi.org/10.1101/2020.10.05.326082

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Molecular Biology
Subject Areas
All Articles
  • Animal Behavior and Cognition (4395)
  • Biochemistry (9613)
  • Bioengineering (7110)
  • Bioinformatics (24914)
  • Biophysics (12642)
  • Cancer Biology (9978)
  • Cell Biology (14377)
  • Clinical Trials (138)
  • Developmental Biology (7967)
  • Ecology (12132)
  • Epidemiology (2067)
  • Evolutionary Biology (16008)
  • Genetics (10937)
  • Genomics (14764)
  • Immunology (9889)
  • Microbiology (23712)
  • Molecular Biology (9492)
  • Neuroscience (50963)
  • Paleontology (370)
  • Pathology (1544)
  • Pharmacology and Toxicology (2688)
  • Physiology (4031)
  • Plant Biology (8677)
  • Scientific Communication and Education (1512)
  • Synthetic Biology (2403)
  • Systems Biology (6446)
  • Zoology (1346)