Defining the purity of exosomes required for diagnostic profiling of small RNA suitable for biomarker discovery
journal contributionposted on 08.04.2022, 06:25 by Camelia Quek, SA Bellingham, CH Jung, Benjamin J Scicluna, Mitch ShambrookMitch Shambrook, Robyn SharplesRobyn Sharples, Lesley SimLesley Sim, Andrew HillAndrew Hill
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. Utilizing an established neuronal cell system, we used next-generation sequencing to comprehensively profile ncRNA in cells and exosomes isolated by these 2 isolation methods. By comparing ncRNA content in exosomes from these two methods, we found that exosomes from both isolation methods were enriched with miRNAs and contained a diverse range of rRNA, small nuclear RNA, small nucleolar RNA and piwi-interacting RNA as compared with 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.
This work was supported by grants from the Australian Research Council (FT100100560 to AFH), the National Health and Medical Research Council (628946 to AFH), Belberry Indigenous Health Fellowships (MDHS, The University of Melbourne) to S.A.B. C.Q. was supported by Melbourne International Research Scholarship.
Pagination14p. (p. 245-258)
PublisherTaylor and Francis
Rights Statement© Camelia Quek, Shayne A. Bellingham, Chol-hee Jung, Benjamin J. Scicluna, Mitch C. Shambrook, Robyn A. Sharples, Lesley Cheng, and Andrew F. Hill. Published with license by Taylor & Francis Group, LLC. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
Science & TechnologyLife Sciences & BiomedicineBiochemistry & Molecular BiologyBiomarkersexosomesmiRNAsmall RNAALZHEIMERS-DISEASENONCODING RNASMALL NUCLEARBIOGENESISMICRORNASFRAGMENTSMIRNAIDENTIFICATIONEXPRESSIONPROGNOSISAnimalsCell LineExosomesGene Expression ProfilingHigh-Throughput Nucleotide SequencingHypothalamusMiceMicroRNAsNeuronsRNA, Small UntranslatedRNA, TransferWorkflowDevelopmental Biology