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.

Figure 1

Both ultracentrifugation protocol and Total exosome isolation reagent enable recovery of very clean population of exosomes from cell media and serum. ((a) and (b)) Analysis of exosomes recovered from HeLa cell media using the Total exosome isolation reagent (from cell culture media) and ultracentrifugation protocol by Nanosight LM10 instrument. ((c) and (d)) Analysis of exosomes recovered from serum using the Total exosome isolation reagent (from serum) and ultracentrifugation protocol by Nanosight LM10 instrument.

Figure 2

Western blot analysis for the presence of exosomal marker proteins CD63 and CD9 in HeLa cell culture media ((a), (b)) and serum ((c), (d)) derived samples. Exosomes isolated with either the Total exosome isolation reagents or ultracentrifugation protocols were separated on 4–20% Tris-Glycine gels. Standard Western blot procedures with anti-CD63 and anti-CD9 antibodies were used to detect exosomal protein markers.

Figure 3

Analysis of the exosomal miRNA levels in HeLa cell culture media (a) and serum preparations (b) by quantitative RT-PCR. RNA was isolated using the Total exosome RNA and protein isolation kit from exosomes extracted using the Total exosome isolation reagents and the ultracentrifugation protocols. Levels of five microRNAs (miR16, miR24, miR26a, miR451, and let7e) were quantified by qRT-PCR using TaqMan assays and reagents.

Figure 4

Iterative mapping pipeline used to map and quantify RNAs sequenced from both exosomes and parental samples. Each read that aligns to a particular RNA is referred to as a “count” of that RNA type. Quantitation is accomplished by calculating a tally of the counts for each type of RNA.

Figure 5

RNA sequencing results for exosomes versus parental HeLa cells. Stacked bar plots depict mapped read counts distributed by RNA species for both HeLa cells and HeLa cell culture media derived exosomes preparations. (a) Full scale. (b) Zooming into 0–10% range on the y-axis to view less prominent RNA types representation.

Figure 6

Differential representation of specific RNA in exosomes versus parental HeLa cells. miRNAs (a), mRNA (b), tRNA (c), and ncRNA (d) from cells and exosome preparations. Quantities were normalized by the percentage of reads aligned to that transcript out of the total mapped.

Figure 7

RNA sequencing results for exosomes versus parental serum. Stacked bar plot depicts mapped read count distribution by RNA species derived from two human blood donors, d1 and d2. Two replicates (rep1, rep2) were sequenced per donor. SnRNA, ScaRNA, SnoRNA, piRNA were also detected though at the very low levels.

Figure 8

Differential representation of specific miRNAs in exosomes versus parental serum samples. Quantities were normalized by the percentage of reads aligned to that transcript out of the total mapped.