title: Characterization and deep sequencing analysis of exosomal and non-exosomal miRNA in human urine.
abstract: Micro RNAs (miRNAs) have been shown to circulate in biological fluids and are enclosed in vesicles such as exosomes; they are present in urine and represent a noninvasive methodology to detect biomarkers for diagnostic testing. The low abundance of RNA in urine creates difficulties in its isolation, of which exosomal miRNA is a small fraction, making downstream RNA assays challenging. Here, we investigate methods to maximize exosomal isolation and RNA yield for next-generation deep sequencing. Upon characterizing exosomal proteins and total RNA content in urine, several commercially available kits were tested for their RNA extraction efficiency. We subsequently used the methods with the highest miRNA content to profile baseline miRNA expression using next-generation deep sequencing. Comparisons of miRNA profiles were also made with exosomes isolated by differential ultracentrifugation methodology and a commercially available column-based protocol. Overall, miRNAs were found to be significantly enriched and intact in urine-derived exosomes compared with cell-free urine. The presence of other noncoding RNAs such as small nuclear and small nucleolar RNA in the exosomes, in addition to coding sequences related to kidney and bladder conditions, was also detected. Our study extensively characterizes the RNA content of exosomes isolated from urine, providing the potential to identify miRNA biomarkers in human urine.
authors.affiliation: 1] Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria, Australia [2] Bio21 Molecular Science and Biotechnology Institute, Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Lesley
authors.initials: L
authors.lastname: Cheng
authors.affiliation: 1] Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria, Australia [2] Bio21 Molecular Science and Biotechnology Institute, Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Xin
authors.initials: X
authors.lastname: Sun
authors.affiliation: 1] Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria, Australia [2] Bio21 Molecular Science and Biotechnology Institute, Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Scicluna
authors.affiliation: 1] Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria, Australia [2] Bio21 Molecular Science and Biotechnology Institute, Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Bradley M
authors.initials: BM
authors.lastname: Coleman
authors.affiliation: 1] Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria, Australia [2] Bio21 Molecular Science and Biotechnology Institute, Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Hill
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000652:urine; D015153:Blotting, Western; D055354:Exosomes / Q000737:chemistry* / Q000235:genetics* / Q000648:ultrastructure; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D005819:Genetic Markers; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000235:genetics* / Q000652:urine*; D046529:Microscopy, Electron, Transmission; D014461:Ultracentrifugation; D016482:Urinalysis / Q000379:methods; D055815:Young Adult
chemical_list: D015415:Biomarkers; D005819:Genetic Markers; D035683:MicroRNAs