Purification of Exosomes from Primary Schwann Cells, RNA Extraction, and Next-Generation Sequencing of Exosomal RNAs

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, researchers from the Universidad Mayor, Chile 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, they 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.

Exosome purification protocol

lncrna

Flowchart showing the main steps for exosome purification from SC conditioned medium. This method is based on serial centrifugation and ultracentrifugation, which eliminates cell debris, microvesicles, and contaminating proteins. Further analysis may be necessary to determine the purity of the final exosome-containing pellet.

De Gregorio C, Díaz P, López-Leal R, Manque P, Court FA. (2018) Purification of Exosomes from Primary Schwann Cells, RNA Extraction, and Next-Generation Sequencing of Exosomal RNAs. Methods Mol Biol 1739:299-315. [abstract]

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