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Comparison of RNA isolation procedures for analysis of adult murine brain and spinal cord astrocytes

Item Type:Article
Title:Comparison of RNA isolation procedures for analysis of adult murine brain and spinal cord astrocytes
Creators Name:Rosiewicz, K.S. and Crowley, T. and Saher, G. and Kerkering, J. and Alisch, M. and Siffrin, V.
Abstract:BACKGROUND: Molecular analyses of cell populations and single cells have been instrumental in the advancement of our understanding of the physiology and pathologic processes of the nervous system. However, the limitation of these methods is the dependence on a gentle, efficient and specific enrichment procedure for the target cell population. In particular, this has been challenging for tightly interconnected cells, for example central nervous system (CNS) endogenous cells such as astrocytes. NEW METHOD: Here we adopted one of the most common methods of cell extraction, namely, enzymatic tissue digestion followed by fluorescence-activated cell sorting (FACS) of individual cells. We evaluated different enzymatic/mechanical tissue dissociation procedures and analyzed different astrocyte lineage transgenic models. Furthermore, we compared the cell extraction efficiency from spinal cord vs. brain. RESULTS: Enzymatic digestion of CNS tissue of Glast-Cre(ERT2)xtdTomato(fl/fl) or Aldh-Cre(ERT2)xtdTomato(fl/fl) followed by FACS resulted in highly purified astrocytes. Automated tissue digestion strongly improved the isolated cell numbers. Aldh1l1-Cre(ERT2) identified more astrocytes than Glast-Cre(ERT2); isolation from brain yields higher numbers than from spinal cord. COMPARISON WITH EXISTING METHODS: We compared the efficiency and purity of the enzymatic dissociation/FACS approach with a more modern procedure consisting of tissue homogenization followed by translating ribosome affinity purification (TRAP). CONCLUSION: We found that both methods result in highly enriched astrocytic RNA. However, only TRAP isolation resulted in reliably detectable RNA concentrations from spinal cord tissue on a single animal level. Depending on the aim of the study both methods have advantages and disadvantages but both are acceptable for astrocytic RNA analysis.
Keywords:Astrocyte, FACS, RNA Analysis, Spinal Cord, Aldh1l1, Glast, ACSA-2, TRAP, Animals
Source:Journal of Neuroscience Methods
ISSN:0165-0270
Publisher:Elsevier (The Netherlands)
Volume:333
Page Range:108545
Date:1 March 2020
Official Publication:https://doi.org/10.1016/j.jneumeth.2019.108545
PubMed:View item in PubMed

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