The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo

Jeoffrey Schageman,Robert Setterquist,Susan Magdaleno,Emily Zeringer,Alexander V Vlassov,Jian Gu,Tim Barta,Kristi Lea,Mu Li

doi:10.1155/2013/253957

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.

Highlights

Exosomes are a type of vesicle, 30–150 nm in size, that have received increased attention over the past few years [1,2,3,4,5,6,7]
Depending on the cell of origin, many different functions have been attributed to exosomes such as morphogen transporters in the creation of polarity during development and differentiation [4], role in programmed cell death, angiogenesis, inflammation, coagulation [9], and migration of Dictyostelium cells by the secretion of chemoattractant signals [10]
We have successfully developed a set of reagents and a complete exosome workflow solution, starting from fast and efficient extraction of exosomes from cell culture media and blood serum to robust isolation of RNA and characterization of exosomal RNA content using Quantitative Real-Time PCR (qRT-PCR) and nextgeneration sequencing methods

Summary

Introduction

Exosomes are a type of vesicle, 30–150 nm in size, that have received increased attention over the past few years [1,2,3,4,5,6,7]. Depending on the cell of origin, many different functions have been attributed to exosomes such as morphogen transporters in the creation of polarity during development and differentiation [4], role in programmed cell death, angiogenesis, inflammation, coagulation [9], and migration of Dictyostelium cells by the secretion of chemoattractant signals [10]. Glioblastoma cells secrete exosomes and microvesicles containing mRNA, miRNA, and angiogenic proteins [12]. From their function in the body to more practical applications, such as the use in diagnostics (based on analysis of their RNA and protein content) and therapeutics development, has grown exponentially in the last five years [18,19,20]

Methods

Results

Conclusion