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Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry
Prasanna Channathodiyil1, Jonathan Houseley1
1Epigenetics Programme, Babraham Institute, Cambridge, United Kingdom.
Plos One
|January 22, 2021
Summary
Researchers developed a new method for extracting high-quality RNA from fixed, stained, and sorted cells using glyoxal instead of formaldehyde. This enables routine transcriptome analysis of purified cell populations and single cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Formaldehyde fixation for flow cytometry impairs RNA extraction and amplification.
- Transcriptome analysis of highly purified cell populations requires high-quality RNA.
Purpose of the Study:
- To develop a method for extracting high-quality RNA from fixed, stained, and sorted cells.
- To enable routine transcriptome analysis of purified cell populations and single cells.
Main Methods:
- Replaced formaldehyde with glyoxal for cell fixation.
- Utilized glyoxal fixation compatible with immunofluorescent staining.
- Performed RNA extraction and RNA-sequencing (RNA-seq) on fixed, stained, and sorted cells.
Main Results:
- Glyoxal fixation preserves RNA integrity and accessibility for enzymatic manipulation.
- RNA extracted from glyoxal-fixed, stained, and sorted cells is suitable for RNA-seq.
- Transcriptomic data from fixed and sorted cells correlate well with data from fresh cells.
Conclusions:
- Glyoxal fixation is a viable alternative to formaldehyde for preserving RNA in flow cytometry.
- This method facilitates routine transcriptome analysis of sorted cells, including single-cell analysis.
