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J Neurosci Methods. 2020 Mar 1;333:108545. doi: 10.1016/j.jneumeth.2019.108545. Epub 2019 Dec 9.

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

Author information

1
Experimental and Clinical Research Center (ECRC), Charité - Universitätsmedizin Berlin und Max Delbrück Center or Molecular Medicine in the Helmholtz Association, Berlin Germany.
2
Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Göttingen Germany.
3
Experimental and Clinical Research Center (ECRC), Charité - Universitätsmedizin Berlin und Max Delbrück Center or Molecular Medicine in the Helmholtz Association, Berlin Germany. Electronic address: siffrinv@gmx.de.

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-CreERT2x tdTomatofl/fl or Aldh1l1-CreERT2x tdTomatofl/fl followed by FACS resulted in highly purified astrocytes. Automated tissue digestion strongly improved the isolated cell numbers. Aldh1l1-CreERT2 identified more astrocytes than Glast-CreERT2; 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:

ACSA-2 - TRAP; Aldh1l1; Astrocyte; FACS; Glast; RNA analysis; Spinal cord

Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no competing interests relevant to this study.

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