Exosome Isolation for Proteomic Analyses and RNA Profiling

Abstract

While the existence of exosomes has been known for over three decades, they have garnered recent interest due to their potential diagnostic and therapeutic relevance. The expression and release of specific tumor-derived proteins into the peripheral circulation has served as the centerpiece of cancer screening and diagnosis. Recently, tissue-associated microRNA (miRNA) has been shown to be characteristic of tumor type and developmental origin, as well as exhibit diagnostic potential. Tumors actively release exosomes, exhibiting proteins and RNAs derived from the originating cell, into the peripheral circulation and other biologic fluids. Recently, we have demonstrated the presence of miRNAs within the RNA fraction of circulating tumor-derived exosomes. Currently, in over 75 investigations compiled in ExoCarta, over 2,300 proteins and 270 miRNAs have been linked with exosomes derived from biologic fluids. Our previous work has indicated that these circulating exosomal proteins and miRNAs can serve as surrogates for the tumor cell-associated counterparts, extending their diagnostic potential to asymptomatic individuals. In this chapter, we compare currently utilized methods for purifying exosomes for postisolation analyses. The exosomes derived from these approaches were assessed for quantity and quality of specific RNA populations and specific marker proteins. These results suggest that, while each method purifies exosomal material, circulating exosomes isolated by ExoQuick precipitation produces exosomal RNA and protein with greater purity and quantity than chromatography, ultracentrifugation, and DynaBeads. While this precipitation approach isolates exosomes in general and does not exhibit specificity for the originating cell, the increased quantity and quality of exosomal proteins and RNA should enhance the sensitivity and accuracy of down-stream analyses, such as qRT-PCR profiling of miRNA and mass spectrometric and electrophoretic analyses of exosomal proteins.