A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue.
Lab Invest
; 102(2): 204-211, 2022 02.
Article
em En
| MEDLINE
| ID: mdl-34775494
ABSTRACT
Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Encéfalo
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RNA
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Núcleo Celular
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Análise de Célula Única
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Células Endoteliais da Veia Umbilical Humana
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Citometria de Fluxo
Tipo de estudo:
Prognostic_studies
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2022
Tipo de documento:
Article