Abstract
Exosomes are small (30–150 nm) vesicles of endosomal origin secreted by most cell types. Exosomes contain proteins, lipids, and RNA species including microRNA, mRNA, rRNA, and long noncoding RNAs. The mechanisms associated with exosome synthesis and cargo loading are still poorly understood. A role for exosomes in intercellular communication has been reported in physiological and pathological conditions both in vitro and in vivo. Previous studies have suggested that Schwann cell-derived exosomes regulate neuronal functions, but the mechanisms are still unclear. Here, we describe protocols to establish rat neonatal Schwann cell cultures and to isolate exosomes from the conditioned medium of these cultures by differential ultracentrifugation. To analyze the RNA content of Schwann cell-derived exosomes, we detail protocols for RNA extraction and next-generation sequencing using miRNA and mRNA libraries. The protocol also includes RNA sequencing of Schwann cells, which allows the comparison between RNA content from cells and the secreted exosomes. Identification of RNAs present in Schwann cell-derived exosomes is a valuable tool to understand novel roles of Schwann cells in neuronal function in health and disease.
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Acknowledgments
We wish to thank all the members of the Court Lab for their contributions to this protocol. This work was supported by the Center for Integrative Biology, Universidad Mayor, FONDECYT-1150766, Geroscience Center for Brain Health and Metabolism (FONDAP-15150012), Ring Initiative ACT1109, and Canada-Israel Health Research Initiative, jointly funded by the Canadian Institutes of Health Research; the Israel Science Foundation; the International Development Research Centre, Canada; and the Azrieli Foundation, Canada.
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De Gregorio, C., Díaz, P., López-Leal, R., Manque, P., Court, F.A. (2018). Purification of Exosomes from Primary Schwann Cells, RNA Extraction, and Next-Generation Sequencing of Exosomal RNAs. In: Monje, P., Kim, H. (eds) Schwann Cells. Methods in Molecular Biology, vol 1739. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7649-2_19
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DOI: https://doi.org/10.1007/978-1-4939-7649-2_19
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