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A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue
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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group US
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8784313/ https://www.ncbi.nlm.nih.gov/pubmed/34775494 http://dx.doi.org/10.1038/s41374-021-00698-z |
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author | Kimble, Amy L. Silva, Jordan Omar, Omar M. Murphy, Melissa Hensel, Jessica A. Nicholas, Sarah-Anne E. Jellison, Evan R. Reese, Bo Murphy, Patrick A. |
author_facet | Kimble, Amy L. Silva, Jordan Omar, Omar M. Murphy, Melissa Hensel, Jessica A. Nicholas, Sarah-Anne E. Jellison, Evan R. Reese, Bo Murphy, Patrick A. |
author_sort | Kimble, Amy L. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8784313 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group US |
record_format | MEDLINE/PubMed |
spelling | pubmed-87843132022-02-04 A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue Kimble, Amy L. Silva, Jordan Omar, Omar M. Murphy, Melissa Hensel, Jessica A. Nicholas, Sarah-Anne E. Jellison, Evan R. Reese, Bo Murphy, Patrick A. Lab Invest Technical Report 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. Nature Publishing Group US 2021-11-13 2022 /pmc/articles/PMC8784313/ /pubmed/34775494 http://dx.doi.org/10.1038/s41374-021-00698-z Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Technical Report Kimble, Amy L. Silva, Jordan Omar, Omar M. Murphy, Melissa Hensel, Jessica A. Nicholas, Sarah-Anne E. Jellison, Evan R. Reese, Bo Murphy, Patrick A. A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue |
title | A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue |
title_full | A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue |
title_fullStr | A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue |
title_full_unstemmed | A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue |
title_short | A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue |
title_sort | method for rapid flow-cytometric isolation of endothelial nuclei and rna from archived frozen brain tissue |
topic | Technical Report |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8784313/ https://www.ncbi.nlm.nih.gov/pubmed/34775494 http://dx.doi.org/10.1038/s41374-021-00698-z |
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