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.
Highlights
High-throughput RNA sequencing (RNA-seq) is increasingly used as a first-pass method to characterise cell populations, providing detailed and robust data through standardised pipelines
We show that glyoxal fixation allows extraction of high-quality RNA using standard protocols from stained and sorted cell samples, and that this RNA can be processed into high quality RNA-seq libraries
RNA extraction was performed using TRI reagent, a monophasic guanidine thiocyanate (GTC)-phenol RNA extraction solution, and 20% of the RNA obtained was analysed on denaturing RNA mini-gels
Summary
High-throughput RNA sequencing (RNA-seq) is increasingly used as a first-pass method to characterise cell populations, providing detailed and robust data through standardised pipelines. Purification of cell populations by flow cytometric cell sorting has become routine, with the capacity to isolate cells based on simultaneous quantification of many antigens. It would seem natural to unite these techniques, such that carefully purified cell populations are characterised by RNA-seq as standard, this has proved surprisingly problematic primarily due to difficulties in RNA recovery from fixed and stained cells. Glyoxal fixation allows transcriptome analysis of sorted cells
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