The purity of exosomes required for diagnostic profiling of small RNA suitable for biomarker discovery

Small non-coding RNAs (ncRNA), including microRNAs (miRNA), enclosed in exosomes are being utilised for biomarker discovery in disease. Two common exosome isolation methods involve differential ultracentrifugation or differential ultracentrifugation coupled with Optiprep gradient fractionation. Generally, the incorporation of an Optiprep gradient provides better separation and increased purity of exosomes. The question of whether increased purity of exosomes is required for small ncRNA profiling, particularly in diagnostic and biomarker purposes, has not been addressed and highly debated.

Utilising an established neuronal cell system, University of Melbourne researchers used Next-Generation sequencing to comprehensively profile ncRNA in cells and exosomes isolated by these two isolation methods. By comparing ncRNA content in exosomes from these two methods, they found that exosomes from both isolation methods were enriched with miRNAs and contained a diverse range of ribosomal RNA, small nuclear RNA, small nucleolar RNA and piwi-interacting RNA as compared to their cellular counterparts. Additionally, tRNA fragments (30-55 nucleotides in length) were identified in exosomes and may act as potential modulators for repressing protein translation. Overall, the outcome of this study confirms that ultracentrifugation-based method as a feasible approach to identify ncRNA biomarkers in exosomes.

Heatmaps of individual small RNA expression profiles

(A) miRNA, (B) piRNA, (C) tRNA, (D) rRNA, (E) snoRNA, (F) snRNA, that are analysed in this study. The majority of the heatmaps depict distinct expression profiles for exosome datasets across all RNA biotypes. The expression profiles for GT1-7 neuronal cells showing distinct profiles among other datasets. The degree of abundance level of different small RNA species is represented by a range of colour starting from blue to yellow to red.