A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue

• Published in: Laboratory investigation 2022-02, Vol.102 (2), p.204-211
• Main Authors: 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.
• Format: Article
• Language: English
• Subjects: 13/31; 38; 38/91; 631/1647/2017/2003; 631/1647/48; 692/699/375/1370; Animal tissues; Animals; Brain; Brain - cytology; Brain - metabolism; Cell Fractionation - methods; Cell Nucleus - metabolism; Cells, Cultured; Cryopreservation - methods; Digestion; Endothelial cells; Endothelium; Flow cytometry; Flow Cytometry - methods; Gene expression; Gene sequencing; HEK293 Cells; Homeostasis; Human tissues; Human Umbilical Vein Endothelial Cells - metabolism; Humans; Laboratory Medicine; Markers; Medicine; Medicine & Public Health; Mice; Mice, Inbred C57BL; Nuclei (cytology); Pathology; Rapid flow; Reproducibility of Results; Ribonucleic acid; RNA; RNA - isolation & purification; RNA - metabolism; Sequence Analysis, RNA - methods; Single-Cell Analysis - methods; Surface markers; technical-report; Tissue Banks; Transcriptional Regulator ERG - metabolism
• ISSN: 0023-6837; 1530-0307
• DOI: 10.1038/s41374-021-00698-z

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.