Microglial FACS for Robust RNA Recovery for Next-Generation Sequencing

Andria L Doty; Aline Oliveria

doi:10.1007/978-1-0716-1783-0_7

Microglial FACS for Robust RNA Recovery for Next-Generation Sequencing

Methods Mol Biol. 2022:2389:77-86. doi: 10.1007/978-1-0716-1783-0_7.

Authors

Andria L Doty¹, Aline Oliveria²

Affiliations

¹ Interdisciplinary Center for Biotechnology, Flow Cytometry & Imaging Core, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA. sun30@ufl.edu.
² Division of Pulmonology, Critical Care and Sleep Medicine, College of Medicine, University of Florida, Gainesville, FL, USA.

PMID: 34558003
DOI: 10.1007/978-1-0716-1783-0_7

Abstract

Microglia are immune cells of the central nervous system (CNS), which play an instrumental role in eliminating invading pathogens and help regulate localized inflammation. They also assist in maintaining homeostasis of the brain microenvironment. Microglia isolation from primary brain tissue can be difficult with poor yields from tissue dissociation which precludes many downstream assays from being efficiently conducted. Recovery of intact microglia for single-cell or next-generation RNA sequencing (NGS, RNAseq) can be a difficult process. The recovery of intact RNA transcripts inside viable cells has its challenges. Here we describe a method to enrich CD11b + microglial cells from brain tissue followed by FACS, for a reliable and reproducible method for the recovery of high-quality RNA from sorted microglia for downstream sequencing.

Keywords: FACS; Microglia; RNA protection and RNA sequencing.

MeSH terms

Brain
Cell Separation
High-Throughput Nucleotide Sequencing
Microglia*
RNA / genetics

Substances

RNA