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

Rosiewicz, K. S., Crowley, T., Saher, G., Kerkering, J., Alisch, M., & Siffrin, V. (2020). Comparison of RNA isolation procedures for analysis of adult murine brain and spinal cord astrocytes. Journal of Neuroscience Methods, 333: 108545. doi:10.1016/j.jneumeth.2019.108545.

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3261042.pdf (Publisher version), 4MB
 
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Rosiewicz, K. S., Author
Crowley, T., Author
Saher, G.1, Author           
Kerkering, J., Author
Alisch, M., Author
Siffrin, V., Author
Affiliations:
1Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, Max Planck Society, ou_3350301              

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Free keywords: Astrocyte FACS RNA analysis Spinal cord Aldh1l1 Glast ACSA-2 - TRAP
 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.

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Language(s): eng - English
 Dates: 2020-03-01
 Publication Status: Published online
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 Rev. Type: Peer
 Identifiers: DOI: 10.1016/j.jneumeth.2019.108545
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Title: Journal of Neuroscience Methods
Source Genre: Journal
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Pages: - Volume / Issue: 333 Sequence Number: 108545 Start / End Page: - Identifier: ISSN: 01650270