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• W2078173329 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. 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. There is increased interest in detecting extracellular microRNA (miRNA) from biological fluids to identify biomarkers for disease. miRNAs are a class of small noncoding RNAs (ncRNAs) that are 22–25 nucleotides long, which function to regulate mRNA processing at the transcriptional and post-transcriptional level. They are derived from mRNA hairpins comprising precursor miRNAs that are further processed by endoribonucleases (Dicer and Drosha) to form mature miRNA. The mature miRNA is incorporated into the RNA-induced silencing complex, which binds to complementary sites in the 3′ untranslated region of their mRNA targets, resulting in the downregulation of gene expression.1.Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29453) Google Scholar miRNA originating from specific tissues can be secreted into the extracellular environment, including biological fluids such as urine.2.Weber J.A. Baxter D.H. Zhang S. et al.The microRNA spectrum in 12 body fluids.Clin Chem. 2010; 56: 1733-1741Crossref PubMed Scopus (1980) Google Scholar Urine is a sterile biological fluid comprising end products generated by protein metabolism that is secreted by the kidneys and can be collected noninvasively and in a simple manner. The majority of urinary miRNA originates from renal and urethral cells, and analysis of these cells can provide a measure of the health of the excretory system.3.Wang G. Kwan B.C. Lai F.M. et al.Urinary miR-21, miR-29, and miR-93: novel biomarkers of fibrosis.Am J Nephrol. 2012; 36: 412-418Crossref PubMed Scopus (124) Google Scholar, 4.Wang G. Kwan B.C. Lai F.M. et al.Expression of microRNAs in the urinary sediment of patients with IgA nephropathy.Dis Markers. 2010; 28: 79-86Crossref PubMed Google Scholar, 5.Cheng L. Quek C.Y. Sun X. et al.The detection of microRNA associated with Alzheimer's disease in biological fluids using next-generation sequencing technologies.Front Genet. 2013; 4: 150Crossref PubMed Scopus (94) Google Scholar Circulating extracellular miRNA from other tissues within the body can be delivered to renal epithelial cells and released into the urine bound to RNA-binding proteins6.Arroyo J.D. Chevillet J.R. Kroh E.M. et al.Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma.Proc Natl Acad Sci USA. 2011; 108: 5003-5008Crossref PubMed Scopus (2495) Google Scholar,7.Vickers K.C. Remaley A.T. Lipid-based carriers of microRNAs and intercellular communication.Curr Opin Lipidol. 2012; 23: 91-97Crossref PubMed Scopus (249) Google Scholar or packaged into microvesicles such as exosomes.8.Valadi H. Ekstrom K. Bossios A. et al.Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat Cell Biol. 2007; 9: 654-659Crossref PubMed Scopus (8870) Google Scholar Exosomes are 40- to 100-nm-diameter vesicles released by cells and are formed within multivesicular bodies in the endosomal system.9.Bellingham S.A. Guo B.B. Coleman B.M. et al.Exosomes: vehicles for the transfer of toxic proteins associated with neurodegenerative diseases?.Front Physiol. 2012; 3: 124Crossref PubMed Scopus (289) Google Scholar They have been found to be enriched in RNA, including coding RNA and ncRNA species such as miRNA, small nuclear RNA, transfer RNA, ribosomal RNA, and long intergenic RNA.8.Valadi H. Ekstrom K. Bossios A. et al.Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat Cell Biol. 2007; 9: 654-659Crossref PubMed Scopus (8870) Google Scholar,10.Bellingham S.A. Coleman B.M. Hill A.F. et al.deep sequencing reveals a distinct miRNA signature released in exosomes from prion-infected neuronal cells.Nucleic Acids Res. 2012; 40: 10937-10949Crossref PubMed Scopus (323) Google Scholar The delivery of large clusters of miRNA in exosomes has the capacity to influence a larger diversity of genes and regulate biological pathways. Profiles of deregulated miRNA secreted into peripheral blood11.Jin P. Wang E. Ren J. et al.Differentiation of two types of mobilized peripheral blood stem cells by microRNA and cDNA expression analysis.J Transl Med. 2008; 6: 39Crossref PubMed Scopus (66) Google Scholar and serum12.Taylor D.D. Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.Gynecol Oncol. 2008; 110: 13-21Abstract Full Text Full Text PDF PubMed Scopus (1940) Google Scholar, 13.Skog J. Wurdinger T. van Rijn S. et al.Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.Nat Cell Biol. 2008; 10: 1470-1476Crossref PubMed Scopus (3698) Google Scholar, 14.Chen X. Ba Y. Ma L. et al.Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases.Cell Res. 2008; 18: 997-1006Crossref PubMed Scopus (3753) Google Scholar have been generated and suggest that they have diagnostic potential for human disease such as gliobastoma and ovarian cancer.12.Taylor D.D. Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.Gynecol Oncol. 2008; 110: 13-21Abstract Full Text Full Text PDF PubMed Scopus (1940) Google Scholar,13.Skog J. Wurdinger T. van Rijn S. et al.Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.Nat Cell Biol. 2008; 10: 1470-1476Crossref PubMed Scopus (3698) Google Scholar Currently, there are limited methodologies available to efficiently isolate extracellular miRNA from the cell-free component of urine. Recent advances in next-generation sequencing (NGS) technologies have allowed the ability to profile total RNA transcriptomes. The challenges faced using NGS to profile small RNA transcriptomes in urine include isolation of total RNA from urine, of which small RNAs make up a fraction, in addition to the added difficulty of isolating exosomal miRNA. Here we have systematically characterized exosomal RNA profiles in human urine. We investigated methodologies to obtain high exosomal yields from minimal urine volumes and tested RNA isolation efficiency by using six commercially available extraction kits in order to maximize RNA yields from these RNA-limited samples. These methods were then analyzed using NGS to profile baseline miRNA and other ncRNAs by small RNA deep sequencing. We present a method that is suitable for analyzing circulating miRNA profiles in urine that can be applied to biomarker discovery studies in diseases affecting the kidney and bladder, such as urothelial and renal cell carcinoma. An optimized differential ultracentrifugation protocol was established to isolate exosomes from urine samples.15.Fernandez-Llama P. Khositseth S. Gonzales P.A. et al.Tamm-Horsfall protein and urinary exosome isolation.Kidney Int. 2010; 77: 736-742Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar,16.Gonzales P.A. Zhou H. Pisitkun T. et al.Isolation and purification of exosomes in urine.Methods Mol Biol. 2010; 641: 89-99Crossref PubMed Scopus (94) Google Scholar Tamm–Horsfall protein (THP) polymerization can lower the yield of exosomes by entrapping exosomes within their protein network.15.Fernandez-Llama P. Khositseth S. Gonzales P.A. et al.Tamm-Horsfall protein and urinary exosome isolation.Kidney Int. 2010; 77: 736-742Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar Dithiothreitol (DTT) has been used in proteomic studies to depolymerize disulphide bonds of THP, releasing entrapped exosomes that pellet upon re-ultracentrifugation and improving the detection of exosomal proteins.15.Fernandez-Llama P. Khositseth S. Gonzales P.A. et al.Tamm-Horsfall protein and urinary exosome isolation.Kidney Int. 2010; 77: 736-742Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar,17.Pisitkun T. Shen R.F. Knepper M.A. Identification and proteomic profiling of exosomes in human urine.Proc Natl Acad Sci USA. 2004; 101: 13368-13373Crossref PubMed Scopus (1623) Google Scholar To investigate the requirement of THP depletion from urine for transcriptomic studies, exosome pellets were treated with or without DTT and re-ultracentrifugation (Figure 1). Exosomes isolated from urine treated with DTT yielded a slight increase of exosomes compared with those treated without DTT, as characterized by western immunoblotting of exosomal markers (Figure 2a). Cell lysates and exosomes from SH-SY5Y cells were used as a positive control given that the methodology to isolate exosomes from cell line supernatant is well established (Figure 2a).18.Vella L.J. Sharples R.A. Lawson V.A. et al.Packaging of prions into exosomes is associated with a novel pathway of PrP processing.J Pathol. 2007; 211: 582-590Crossref PubMed Scopus (339) Google Scholar,19.Sharples R.A. Vella L.J. Nisbet R.M. et al.Inhibition of gamma-secretase causes increased secretion of amyloid precursor protein C-terminal fragments in association with exosomes.FASEB J. 2008; 22: 1469-1478Crossref PubMed Scopus (214) Google Scholar Furthermore, no significant difference was observed upon comparing the yield of RNA content extracted from exosomes treated with or without DTT (Figure 2b). To directly visualize the exosomes, transmission electron microscopy (TEM) was used. Vesicles isolated by differential ultracentrifugation were found to have a diameter of 50–100nm and morphology consistent with previous reports (Figure 2c).20.Coleman B.M. Hanssen E. Lawson V.A. et al.Prion-infected cells regulate the release of exosomes with distinct ultrastructural features.FASEB J. 2012; 26: 4160-4173Crossref PubMed Scopus (112) Google Scholar,21.Thery C. Zitvogel L. Amigorena S. Exosomes: composition, biogenesis and function.Nat Rev Immunol. 2002; 2: 569-579Crossref PubMed Scopus (3741) Google Scholar Treatment with DTT significantly reduced the presence of the THP fibril network, as observed in the TEM images (Figure 2c). We further characterized the sizes of vesicles from cell-free urine and vesicles isolated by ultracentrifugation via the particle-to-particle qNano nanopore-based instrument. The majority of vesicle particles were also found to be between 50 and 100nm in diameter (Figure 2d). Given that the treatment with DTT did not significantly improve exosomal yields (Figure 2a) or affect RNA extraction (Figure 2b), we considered this treatment to be unnecessary for this transcriptomic study. Further characterization of exosomes isolated without DTT treatment by western immunoblotting of exosomal markers (Figure 3a) and non-exosomal markers (nucleoporin, GM130, and Bcl-2) were observed to be highly enriched upon differential ultracentrifugation compared with the various fractions collected during the procedure (whole urine, cell pellet, cell-free urine, and exosome-depleted supernatant, Figure 3a). In addition, upon analyzing the small RNA species (<200nt) extracted from individual components of urine, small RNA was found to be enriched in the exosomal pellets (Figure 3b). Although the cell pellet obtained from whole urine contained significant amounts of small RNA, the majority was found to be degraded RNA (Figure 3c). Large RNA species (>200nt), in particular 18S and 28S ribosomal RNA, were not detected in the cell pellet, as determined by Bioanalyser analysis using an RNA Nano chip (RNA integrity number=0, Figure 3c), suggesting degradation of the cellular RNA. A systematic comparison between different RNA extraction methods was performed on urine samples uniformly pooled from four healthy control subjects (Supplementary Table S1 online) to obtain sufficient volume for three independent experiments (Figure 4). Six commercial RNA isolation kits were tested using the pooled urine samples. The kits compared in this section were miRNeasy (Qiagen), miRNeasy with RNeasy MinElute Cleanup Kit (Qiagen), mirVana PARIS (Ambion), Trizol LS reagent (Life Technologies) with mirVana (Ambion), miRCURY (Exiqon), and the Urine Exosome RNA Isolation kit (Norgen Biotek). Several modifications of RNA extraction were investigated, including phenol/chloroform extraction and further miRNA enrichment, to determine the best methodology for maximal RNA yield. Download .doc (.03 MB) Help with doc files Supplementary Table 1 With the exception of the Norgen Urine Exosome Isolation kit, the remaining kits involve the isolation of exosomes by differential ultracentrifugation followed by RNA extraction. The majority of these kits use guanidine and phenol extraction methods owing to their combined high efficiency in separating protein and RNA into an interphase and aqueous phase, respectively. One exception is the mirVana kit (Life Technologies), which supplies a phenol-free lysis reagent. Therefore, a minor modification of the mirVana kit was also tested whereby the manufacturer’s lysis/binding reagent was replaced with Trizol LS reagent. In addition, enrichment and separation of miRNA (18–200nt) from total RNA (>200nt) was also investigated by using the miRNeasy kit and the RNeasy MinElute Cleanup kit. The Urine Exosome RNA Isolation kit from Norgen Biotek is a centrifugation-independent kit that isolates exosomes by binding urinary exosomes to a proprietary resin and enriching exosomal RNA by lysing the bound exosomes. The RNA is released from the exosomes, which is transferred to a column and contaminants are then removed by washes before the elution of exosomal RNA. We evaluated the Norgen Biotek kit to determine whether a similar result could be achieved for deep sequencing without using the lengthy ultracentrifugation procedure. RNA extractions were analyzed using a Bioanalyser with the small RNA assays normalized to ng miRNA per ml of urine (Figure 5). The Norgen Biotek kit isolated more than a fourfold increase of small RNA compared with the other isolation kits (P<0.001, Figure 5a). Among the five kits dependent on the use of differential ultracentrifugation to isolate exosomes, similar yields of small RNA were obtained. Regardless of the kit used, there was high reproducibility and consistency of RNA yield. To confirm an enrichment of intact miRNA in exosomes, RNA extraction was also performed on the cell pellet (Figure 5b) and the cell-free component of urine (Figure 5c). As expected, there was less consistency and miRNA extracted from the cell pellet, as this contains mostly cellular debris and degraded RNA (Figure 3c), although most kits were able to extract nucleic acids of less than 200nt. Overall, a higher yield of total small RNA was extracted from cell-free urine compared with the pellet of isolated exosomes (Figure 5c). This is not surprising, as the cell-free urine analyzed in Figure 5c had not been depleted of exosomes. It is important to note that the integrity of the small RNA isolated in cell-free urine cannot be analyzed owing to the absence of ribosomal RNA. Deep sequencing would provide an insight into whether there is an intact population of miRNA fragments extracted from cell-free urine. In addition to the amount of RNA obtained per sample, the percentage of miRNA recovered from the total RNA extracted was also analyzed by using a Bioanalyser small RNA assay. The highest percentage of miRNA was extracted from exosomes compared with the cell pellet and cell-free urine, suggesting that there is an enrichment of miRNA in exosomes. The most efficient kit that isolated the highest percentage of miRNA was the miRNeasy kit through the separation and enrichment of miRNA from large RNA using the additional RNeasy MinElute column (80%). The second most efficient was the miRNeasy kit (without miRNA enrichment; 70%), followed by the other three kits (approximately 60%). Although miRNA comprised 50% of the small RNA extracted by the Norgen kit, overall the miRNA yield was considerably higher compared with the other methods of extraction (summarized in Table 1). Although considerable percentages of miRNA were obtained from the cell pellet and cell-free urine, it cannot be determined whether these fragments between 10 and 40nt are miRNA or degraded RNA.Table 1Performance of six commercial RNA extraction kits in extracting RNA from urinary exosomesKitUCSample volume (ml)Time (h)Efficiency% miRNANorgenNo5–101.5Very highMediummiRNeasyYes⩾204HighHighmiRNeasy-RNeasy4.5MediumVery highmirVana4MediumMediumTrizol LS + mirVana4.5HighMediummiRCURY4MediumMediumAbbreviations: miRNA, micro RNA; UC, ultracentrifuge. Open table in a new tab Abbreviations: miRNA, micro RNA; UC, ultracentrifuge. On comparing preparation time of the differential ultracentrifugation procedure (4h), it was found that the Norgen Biotek kit eliminates almost 3h of preparation time and uses minimal sample volume (5ml) to isolate exosomal RNA for downstream assays (Table 1). Unfortunately, protein characterization of the exosomes cannot be performed with the samples extracted from the Norgen Biotek kit. Owing to the high efficiency of the Norgen Biotek kit, we decided to continue the use of this kit to profile exosomal miRNA by deep sequencing and compare the miRNA profiles with exosomes isolated using the differential ultracentrifugation method. Urine samples from three different subjects (Supplementary Table S1 online) were sequenced using the Ion Torrent Personal Genome Machine (Ion PGM, Life Technologies) sequencing platform. Small RNA libraries were constructed using RNA extracted from exosomes isolated from the ultracentrifugation method and the Norgen Biotek kit. To observe whether there was a selective profile of exosomal miRNA and enrichment by using these methods, deep sequencing was also performed on cell-free urine. Deep sequencing was not performed on cell pellets, as the small RNA libraries constructed were found to contain a high abundance of small complementary DNA (cDNA) inserts, most likely of degraded RNA. miRNA enrichment using the RNeasy MinElute column was not performed, as cDNA library preparation for deep sequencing involves size selection of small RNA, and hence the additional miRNA enrichment step was unnecessary. In addition, the majority of RNA species contained in the exosomes were small RNAs. In all, nine libraries were constructed (n=3) with identifiable indexes and sequenced on three 318 Ion Torrent chips using the Ion PGM platform. A total of 7,075,206 reads were obtained with an average of 786,134 single-end reads produced per sample. Raw sequences were aligned to the human genome (HG19), and reads were mapped to miRBase V.1922.Griffiths-Jones S. The microRNA Registry.Nucleic Acids Res. 2004; 32: D109-D111Crossref PubMed Google Scholar (Figure 6). Of these, 535 known miRNAs were identified, which comprised 35% of total reads (Figure 7a, Supplementary Data SD1 online). Upon removing sequences mapped to miRNA, the remaining sequences were mapped to other ncRNAs of the human genome using Ensembl annotations (Homo_sapiens.GRCh37.72).23.Flicek P. Ahmed I. Amode M.R. et al.Ensembl 2013.Nucleic Acids Res. 2013; 41: D48-D55Crossref PubMed Scopus (796) Google Scholar Less than 1% was found to map to other ncRNAs such as small nuclear RNA (0.02%), snoRNA (0.04%), LncRNA (0.02%), and LinRNA (0.17%; Figure 7a). Coding RNA (3%) mapped to the human genome was also detected in all samples (Supplementary Table 1, Supplementary Table 2). Download .xls (.98 MB) Help with xls files Supplementary Table 2 Low abundant miRNA sequences containing less than five normalized reads per million were removed. The removal of low abundant miRNA reads eliminates possible artifacts obtained from the normalization of miRNA that are not present in one or more samples. Therefore, miRNA with high read counts are considered abundant and consist of actual raw reads before and after post-processing of NGS data. From our analyses, only 12 miRNAs were abundantly expressed in cell-free urine compared with 66 and 184 miRNAs detected in RNA isolated using the Norgen Biotek kit and by ultracentrifugation of exosomes, respectively (Figure 7b, Supplementary Data SD4 online). For each miRNA identified, the majority of miRNA was present in all three subjects (Figure 8). Upon analyzing common and unique miRNA across all samples, seven miRNAs were found to be common in all samples (Figure 7b). Surprisingly, there was a low abundance of miRNA in cell-free urine. Although a high yield of small RNA was extracted from cell-free urine (Figure 5c), it is possible that non-exosomal RNA circulating in cell-free urine is substantially degraded and consequently does not map to miRNA annotations in miRBase. Only two miRNAs (hsa-miR-3648 and hsa-miR-4516) were found to be specifically detected in cell-free urine. A total of 182 miRNAs identified were exosomal specific. By comparing the two exosome preparations, 5 miRNAs were specifically isolated using the Norgen Biotek kit (Table 2) and 51 miRNAs were detected in both samples.Table 2Exosomal-specific miRNA detected in UC and NG exosome samplesNG exosomesReads (r.p.m.)UCaRNA extracted by the miRNeasy kit. and NG exosomesUC reads (r.p.m.)NG reads (r.p.m.)UC exosomesaRNA extracted by the miRNeasy kit.Reads (r.p.m.)hsa-miR-124-3p6hsa-miR-30a-5p313594hsa-miR-29c-3p560hsa-miR-145-5p5hsa-miR-30b-5p413583hsa-miR-200a-3p510hsa-miR-323b-5p9hsa-miR-10b-5p533041hsa-let-7e-5p453hsa-miR-44975hsa-miR-10a-5p302076hsa-miR-141-3p345hsa-miR-486-5p7hsa-miR-30d-5p131568hsa-miR-429218hsa-miR-26a-5p191467hsa-let-7d-5p182hsa-miR-30c-5p211341hsa-miR-20a-5p174hsa-miR-200c-3p281180hsa-miR-26b-5p170hsa-miR-99a-5p54792hsa-miR-17-5p169hsa-let-7b-5p62779hsa-miR-30e-5p157hsa-miR-19b-3p13718hsa-miR-93-5p155hsa-miR-200b-3p8703hsa-miR-27b-3p155hsa-let-7a-5p7609hsa-miR-27a-3p139hsa-miR-29b-3p21476hsa-miR-660-5p136hsa-miR-203a39454hsa-miR-221-3p129hsa-miR-21-5p16446hsa-miR-30a-3p119hsa-let-7f-5p7425hsa-miR-19a-3p108hsa-let-7g-5p8358hsa-miR-103a-3p105hsa-miR-29a-3p9310hsa-miR-103b105hsa-miR-125b-5p29243hsa-miR-23a-3p103hsa-let-7c10201hsa-miR-135b-5p97hsa-miR-23b-3p11200hsa-miR-424-5p94hsa-miR-24-3p7174hsa-miR-374b-5p84hsa-miR-99b-5p8170hsa-miR-374c-3p84hsa-miR-335-5p7139hsa-miR-106a-5p76Abbreviations: miRNA, micro RNA; NG, Norgen Biotek Urine Exosomal RNA kit; UC, ultracentrifuge.All miRNAs identified and accession numbers in Supplementary Data SD6 online.Data uploaded on Vesiclepedia (http://www.microvesicles.org/exp_summary?exp_id=357).a RNA extracted by the miRNeasy kit. Open table in a new tab Abbreviations: miRNA, micro RNA; NG, Norgen Biotek Urine Exosomal RNA kit; UC, ultracentrifuge. All miRNAs identified and accession numbers in Supplementary Data SD6 online. Data uploaded on Vesiclepedia (http://www.microvesicles.org/exp_summary?exp_id=357). However, the majority of miRNAs (184) identified in this study were extracted from exosomes isolated by using the ultracentrifuge, comprising 126 specific miRNAs (Figure 7b). The 10 most abundant miRNAs detected in the three samples (n=3) were pooled and presented in Table 3. Those that are common across the three samples are outlined in bold. This table illustrates that the Norgen Biotek kit isolates miRNA that can be found both in cell-free and ultracentrifuge exosomes.Table 3The 10 most highly expressed miRNACell-free urineaRNA extracted by the miRNeasy kit.Reads (r.p.m.)NG exosomesReads (r.p.m.)UC exosomesaRNA extracted by the miRNeasy kit.Reads (r.p.m.)hsa-miR-608751hsa-miR-320a197hsa-miR-30a-5p3594hsa-miR-451a22hsa-miR-125a-5p100hsa-miR-30b-5p3583hsa-miR-223-3p17hsa-miR-451a68hsa-miR-10b-5p3041hsa-miR-191-5p15hsa-miR-191-5p67hsa-miR-10a-5p2076hsa-miR-364815hsa-let-7b-5p62hsa-miR-30d-5p1568hsa-miR-44889hsa-miR-223-3p61hsa-miR-26a-5p1503hsa-miR-204-5p9hsa-miR-192-5p55hsa-miR-30c-5p1343hsa-miR-125a-5p7hsa-miR-34a-5p55hsa-miR-200c-3p1180hsa-miR-1347hsa-miR-99a-5p54hsa-miR-99a-5p792hsa-miR-16-5p7hsa-miR-10b-5p53hsa-let-7b-5p779Abbreviations: miRNA, micro RNA; NG, Norgen Biotek Urine Exosomal RNA kit; UC, ultracentrifuge.Bold font indicates common miRNA.All miRNAs identified and accession numbers in Supplementary Data SD7 online.a RNA extracted by the miRNeasy kit. Open table in a new tab Abbreviations: miRNA, micro RNA; NG, Norgen Biotek Urine Exosomal RNA kit; UC, ultracentrifuge. Bold font indicates common miRNA. All miRNAs identified and accession numbers in Supplementary Data SD7 online. The advantages of using urine in clinical tests are that it is collected noninvasively, and the procedure is relatively fast and cost-efficient compared with other clinical samples such as blood and cerebrospinal fluid. A number of recent studies have pooled patient samples in order to perform microarray or quantitative PCR analysis, as it is difficult to obtain enough RNA to successfully construct cDNA libraries for high-throughput deep sequencing.24.Alvarez M.L. Khosroheidari M. Kanchi Ravi R. et al.Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers.Kidney Int. 2012; 82: 1024-1032Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar,25.Gildea J.J. Carlson J.M. Schoeffel C.D. et al.Urinary exosome miRNome analysis and its applications to salt sensitivity of blood pressure.Clin Biochem. 2013: 1131-1134Crossref Scopus (58) Google Scholar Most studies use the urine cellular sediment obtained after low-speed centrifugation for miRNA analysis.4.Wang G. Kwan B.C. Lai F.M. et al.Expression of microRNAs in the urinary sediment of patients with IgA nephropathy.Dis Markers. 2010; 28: 79-86Crossref PubMed Google Scholar,26.Bryant R.J. Pawlowski T. Catto J.W. et al.Changes in circulating microRNA levels associated with prostate cancer.Br J Cancer. 2012; 106: 768-774Crossref PubMed Scopus (571) Google Scholar, 27.Snowdon J. Boag S. 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• W2078173329 created "2016-06-24" @default.
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• W2078173329 date "2014-08-01" @default.
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• W2078173329 title "Characterization and deep sequencing analysis of exosomal and non-exosomal miRNA in human urine" @default.
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