title: Methods for the extraction and RNA profiling of exosomes.
abstract: OBJECTIVE
  To develop protocols for isolation of exosomes and characterization of their RNA content.
  
  
  METHODS
  Exosomes were extracted from HeLa cell culture media and human blood serum using the Total exosome isolation (from cell culture media) reagent, and Total exosome isolation (from serum) reagent respectively. Identity and purity of the exosomes was confirmed by Nanosight(®) analysis, electron microscopy, and Western blots for CD63 marker. Exosomal RNA cargo was recovered with the Total exosome RNA and protein isolation kit. Finally, RNA was profiled using Bioanalyzer and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) methodology.
  
  
  RESULTS
  Here we describe a novel approach for robust and scalable isolation of exosomes from cell culture media and serum, with subsequent isolation and analysis of RNA residing within these vesicles. The isolation procedure is completed in a fraction of the time, compared to the current standard protocols utilizing ultracentrifugation, and allows to recover fully intact exosomes in higher yields. Exosomes were found to contain a very diverse RNA cargo, primarily short sequences 20-200 nt (such as miRNA and fragments of mRNA), however longer RNA species were detected as well, including full-length 18S and 28S rRNA.
  
  
  CONCLUSIONS
  We have successfully developed a set of reagents and a workflow allowing fast and efficient extraction of exosomes, followed by isolation of RNA and its analysis by qRT-PCR and other techniques.
authors.affiliation: Emily Zeringer, Mu Li, Tim Barta, Jeoffrey Schageman, Ketil Winther Pedersen, Axl Neurauter, Susan Magdaleno, Robert Setterquist, Alexander V Vlassov, Life Technologies, Austin, TX 78744, United States.
authors.forename: Emily
authors.initials: E
authors.lastname: Zeringer
authors.affiliation: Emily Zeringer, Mu Li, Tim Barta, Jeoffrey Schageman, Ketil Winther Pedersen, Axl Neurauter, Susan Magdaleno, Robert Setterquist, Alexander V Vlassov, Life Technologies, Austin, TX 78744, United States.
authors.forename: Mu
authors.initials: M
authors.lastname: Li
authors.affiliation: Emily Zeringer, Mu Li, Tim Barta, Jeoffrey Schageman, Ketil Winther Pedersen, Axl Neurauter, Susan Magdaleno, Robert Setterquist, Alexander V Vlassov, Life Technologies, Austin, TX 78744, United States.
authors.forename: Tim
authors.initials: T
authors.lastname: Barta
authors.affiliation: Emily Zeringer, Mu Li, Tim Barta, Jeoffrey Schageman, Ketil Winther Pedersen, Axl Neurauter, Susan Magdaleno, Robert Setterquist, Alexander V Vlassov, Life Technologies, Austin, TX 78744, United States.
authors.forename: Jeoffrey
authors.initials: J
authors.lastname: Schageman
authors.affiliation: Emily Zeringer, Mu Li, Tim Barta, Jeoffrey Schageman, Ketil Winther Pedersen, Axl Neurauter, Susan Magdaleno, Robert Setterquist, Alexander V Vlassov, Life Technologies, Austin, TX 78744, United States.
authors.forename: Ketil Winther
authors.initials: KW
authors.lastname: Pedersen
authors.affiliation: Emily Zeringer, Mu Li, Tim Barta, Jeoffrey Schageman, Ketil Winther Pedersen, Axl Neurauter, Susan Magdaleno, Robert Setterquist, Alexander V Vlassov, Life Technologies, Austin, TX 78744, United States.
authors.forename: Axl
authors.initials: A
authors.lastname: Neurauter
authors.affiliation: Emily Zeringer, Mu Li, Tim Barta, Jeoffrey Schageman, Ketil Winther Pedersen, Axl Neurauter, Susan Magdaleno, Robert Setterquist, Alexander V Vlassov, Life Technologies, Austin, TX 78744, United States.
authors.forename: Susan
authors.initials: S
authors.lastname: Magdaleno
authors.affiliation: Emily Zeringer, Mu Li, Tim Barta, Jeoffrey Schageman, Ketil Winther Pedersen, Axl Neurauter, Susan Magdaleno, Robert Setterquist, Alexander V Vlassov, Life Technologies, Austin, TX 78744, United States.
authors.forename: Robert
authors.initials: R
authors.lastname: Setterquist
authors.affiliation: Emily Zeringer, Mu Li, Tim Barta, Jeoffrey Schageman, Ketil Winther Pedersen, Axl Neurauter, Susan Magdaleno, Robert Setterquist, Alexander V Vlassov, Life Technologies, Austin, TX 78744, United States.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Vlassov
keywords: Cell culture media; Exosomes; Microvesicles; Quantitative reverse transcription-polymerase chain reaction; RNA; Sequencing; Serum