title: The complete exosome workflow solution: from isolation to characterization of RNA cargo.
abstract: Exosomes are small (30-150 nm) vesicles containing unique RNA and protein cargo, secreted by all cell types in culture. They are also found in abundance in body fluids including blood, saliva, and urine. At the moment, the mechanism of exosome formation, the makeup of the cargo, biological pathways, and resulting functions are incompletely understood. One of their most intriguing roles is intercellular communication--exosomes function as the messengers, delivering various effector or signaling macromolecules between specific cells. There is an exponentially growing need to dissect structure and the function of exosomes and utilize them for development of minimally invasive diagnostics and therapeutics. Critical to further our understanding of exosomes is the development of reagents, tools, and protocols for their isolation, characterization, and analysis of their RNA and protein contents. Here we describe a complete exosome workflow solution, starting from fast and efficient extraction of exosomes from cell culture media and serum to isolation of RNA followed by characterization of exosomal RNA content using qRT-PCR and next-generation sequencing techniques. Effectiveness of this workflow is exemplified by analysis of the RNA content of exosomes derived from HeLa cell culture media and human serum, using Ion Torrent PGM as a sequencing platform.
authors.affiliation: Life Technologies, Austin, TX 78744, USA.
authors.forename: Jeoffrey
authors.initials: J
authors.lastname: Schageman
authors.forename: Emily
authors.initials: E
authors.lastname: Zeringer
authors.forename: Mu
authors.initials: M
authors.lastname: Li
authors.forename: Tim
authors.initials: T
authors.lastname: Barta
authors.forename: Kristi
authors.initials: K
authors.lastname: Lea
authors.forename: Jian
authors.initials: J
authors.lastname: Gu
authors.forename: Susan
authors.initials: S
authors.lastname: Magdaleno
authors.forename: Robert
authors.initials: R
authors.lastname: Setterquist
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Vlassov
mesh_terms: D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D003470:Culture Media; D055354:Exosomes / Q000378:metabolism*; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000097:blood / Q000235:genetics; D012313:RNA / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D044967:Serum / Q000378:metabolism; D060245:Tetraspanin 29 / Q000378:metabolism; D060149:Tetraspanin 30 / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D003470:Culture Media; D035683:MicroRNAs; D060245:Tetraspanin 29; D060149:Tetraspanin 30; D012313:RNA