title: Defining the purity of exosomes required for diagnostic profiling of small RNA suitable for biomarker discovery.
abstract: 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.
authors.affiliation: a Department of Biochemistry and Molecular Biology , Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Melbourne , VIC , Australia.|b Department of Biochemistry and Genetics , La Trobe Institute for Molecular Science, La Trobe University , VIC , Australia.
authors.forename: Camelia
authors.initials: C
authors.lastname: Quek
authors.affiliation: a Department of Biochemistry and Molecular Biology , Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Melbourne , VIC , Australia.
authors.forename: Shayne A
authors.initials: SA
authors.lastname: Bellingham
authors.affiliation: c VLSCI Life Sciences Computation Centre, University of Melbourne , VIC , Australia.
authors.forename: Chol-Hee
authors.initials: CH
authors.lastname: Jung
authors.affiliation: a Department of Biochemistry and Molecular Biology , Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Melbourne , VIC , Australia.|b Department of Biochemistry and Genetics , La Trobe Institute for Molecular Science, La Trobe University , VIC , Australia.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Scicluna
authors.affiliation: b Department of Biochemistry and Genetics , La Trobe Institute for Molecular Science, La Trobe University , VIC , Australia.
authors.forename: Mitch C
authors.initials: MC
authors.lastname: Shambrook
authors.affiliation: a Department of Biochemistry and Molecular Biology , Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Melbourne , VIC , Australia.
authors.forename: Robyn A
authors.initials: RA
authors.lastname: Sharples
authors.affiliation: b Department of Biochemistry and Genetics , La Trobe Institute for Molecular Science, La Trobe University , VIC , Australia.
authors.forename: Lesley
authors.initials: L
authors.lastname: Cheng
authors.affiliation: b Department of Biochemistry and Genetics , La Trobe Institute for Molecular Science, La Trobe University , VIC , Australia.
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Hill
mesh_terms: D000818:Animals; D015415:Biomarkers; D002460:Cell Line; D055354:Exosomes / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing; D007031:Hypothalamus / Q000166:cytology / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000235:genetics; D009474:Neurons / Q000378:metabolism; D058727:RNA, Small Untranslated / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics; D057188:Workflow
chemical_list: D015415:Biomarkers; D035683:MicroRNAs; D058727:RNA, Small Untranslated; D012343:RNA, Transfer
keywords: Biomarkers; exosomes; miRNA; small RNA