Brief Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Methodol. Mar 26, 2013; 3(1): 11-18
Published online Mar 26, 2013. doi: 10.5662/wjm.v3.i1.11
Methods for the extraction and RNA profiling of exosomes
Emily Zeringer, Mu Li, Tim Barta, Jeoffrey Schageman, Ketil Winther Pedersen, Axl Neurauter, Susan Magdaleno, Robert Setterquist, Alexander V Vlassov
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
Author contributions: Zeringer E, Li M, Barta T, Schageman J, Pedersen KW and Neurauter A were involved in design of the study, data acquisition, analysis and interpretation of data; Magdaleno S, Setterquist R and Vlassov AV contributed to the conception and design of the study, and wrote the article.
Correspondence to: Alexander V Vlassov, PhD, Life Technologies, 2130 Woodward street, Austin, TX 78744, United States. sasha.vlassov@lifetech.com
Telephone: +1-512-7213843 Fax: +1-512-6510201
Received: February 27, 2013
Revised: March 8, 2013
Accepted: March 18, 2013
Published online: March 26, 2013
Abstract

AIM: 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.

CONCLUSION: 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.

Keywords: Exosomes, Microvesicles, Cell culture media, Serum, RNA, Quantitative reverse transcription-polymerase chain reaction, Sequencing

Core tip: Exosomes are small vesicles (30-150 nm) perceived to be carriers of the unique effector or signaling macromolecules (miRNA, ncRNA, mRNA and protein) between very specific cells within our body. The spectrum of current scientific interests ranges from studying the functions and pathways of exosomes to utilizing them in diagnostics and therapeutics development. Here we describe a complete exosome workflow solution: fast and efficient isolation of exosomes; extraction of their cargo; characterization of exosomal RNA content using quantitative reverse transcription-polymerase chain reaction and other techniques.