A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue.
A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue.
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DOI：
10.1038/s41374-021-00698-z
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发表时间：
2022-03
期刊：
影响因子：
--
通讯作者：
Murphy PA
中科院分区：
文献类型：
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作者：
Kimble AL;Silva J;Omar OM;Murphy M;Hensel JA;Nicholas SE;Jellison ER;Reese B;Murphy PA
内皮细胞是大脑发育，生理和疾病的重要贡献者。虽然RNA测序有助于理解脑内皮细胞的多样性，批量分析和单细胞方法依赖于新鲜组织消化协议的分离单个内皮细胞和基于流式细胞术的表面标记或转基因表达的分选。这些方法在人脑组织中的内皮分析中受到限制，其中难以获得新鲜样品。在这里，我们开发了一种方法来检查内皮RNA的表达，通过使用内皮特异性标记物，从大量存档的冷冻脑组织中分离细胞核。我们表明，这种方法快速，可靠地提取内皮细胞核从冷冻小鼠脑样品，重要的是，从存档的冷冻人脑组织。此外，分离的RNA转录物水平与来自组织消化方案的全细胞中的表达密切相关，并且富含内皮标志物并且耗尽其他脑细胞类型的标志物。由于高质量的RNA转录本可以从存档的冷冻人脑组织中的少至100个细胞核中获得，我们预测这种方法对于人脑组织中内皮RNA转录本的批量分析以及内皮亚群的单细胞分析都是有用的。从存档的人类标本中特异性分离这些细胞的方法将加速对内皮功能的理解。在这里，作者使用内皮转录因子Erg从小鼠和人体组织中分离细胞核，为高通量表征内皮在稳态和疾病中的功能铺平了道路。
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. Understanding of endothelial functions would be accelerated by methods for the specific isolation of these cells from archived human specimens. Here, the authors use the endothelial transcription factor Erg to isolate nuclei from mouse and human tissues, paving the way for high-throughput characterization of the function of endothelium in homeostasis and disease.