Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers

Abstract

Exosomes are tiny vesicles (30-150 nm) constantly secreted by all healthy and abnormal cells, and found in abundance in all body fluids. These vesicles, loaded with unique RNA and protein cargo, have a wide range of biological functions, including cell-to-cell communication and signalling. As such, exosomes hold tremendous potential as biomarkers and could lead to the development of minimally invasive diagnostics and next generation therapies within the next few years. Here, we describe the strategies for isolation of exosomes from human blood serum and urine, characterization of their RNA cargo by sequencing, and present the initial data on exosome labelling and uptake tracing in a cell culture model. The value of exosomes for clinical applications is discussed with an emphasis on their potential for diagnosing and treating neurodegenerative diseases and brain cancer.

Keywords: biomarker; circulating RNA; exosomes; extracellular vesicles.

Figure 1.

Size distribution and concentration of exosomes recovered from urine (a) and serum (b), using their specific Total exosome isolation reagents and protocol. Analysis performed on the NanoSight LM10 instrument.

Figure 2.

Sequencing results for exosomes isolated from human serum and urine. Two donors were used for each sample type (D1 and D2), and for each donor two libraries were prepared and sequenced individually. The PGM 318 chips hold over 11 million wells. For the samples derived from serum as well as urine, 9–10 million wells were loaded (75–90%) and sequenced. After subtracting the polyclonals, low-quality sequences and non-templated ion sphere particles, 5–6 million final readouts were obtained from each run. Of those reads, 90–98% was mapped following the earlier developed pipeline [16].

Figure 3.

Heatmap with dendrogram depicting clustering results of miRNA representation across urine and serum samples types. Two donors were used for each sample type, and for each donor two technical replicates were performed. A hierarchical clustering was applied using the average linkage method and Euclidean distance. Values for the heatmap represent the log2-transformed fraction of total mapped reads being allocated to each miRNA per library. Light-red shaded boxes indicate potential miRNA markers described in the analysis section.

Figure 4.

Uptake by HeLa cells of exosomes labelled with SYTO RNASelect stain. A FLoid Cell Imaging station was used. Red: Alexa Fluor 594; blue: DAPI; green: SYTO RNASelect stain. (a) Labelled exosomes added to cells, (b) dye only control and (c) no treatment control (cells only).