Comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease
Urinary Extracellular Vesicles (uEV) have emerged as a source for biomarkers of kidney damage, holding potential to replace the conventional invasive techniques including kidney biopsy. However, comprehensive studies characterizing uEV isolation methods with patient samples are rare. Here we compare...
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| Veröffentlicht in: | Journal of extracellular vesicles 2020-12, Vol.10 (2), p.e12038-n/a |
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| creator | Barreiro, Karina Dwivedi, Om Prakash Leparc, German Rolser, Marcel Delic, Denis Forsblom, Carol Groop, Per‐Henrik Groop, Leif Huber, Tobias B. Puhka, Maija Holthofer, Harry |
| description | Urinary Extracellular Vesicles (uEV) have emerged as a source for biomarkers of kidney damage, holding potential to replace the conventional invasive techniques including kidney biopsy. However, comprehensive studies characterizing uEV isolation methods with patient samples are rare. Here we compared performance of three established uEV isolation workflows for their subsequent use in transcriptomics analysis for biomarker discovery in diabetic kidney disease. We collected urine samples from individuals with type 1 diabetes with macroalbuminuria and healthy controls. We isolated uEV by Hydrostatic Filtration Dialysis (HFD), ultracentrifugation (UC), and a commercial kit‐ based isolation method (NG), each with different established urine clearing steps. Purified EVs were analysed by electron microscopy, nanoparticle tracking analysis, and Western blotting. Isolated RNAs were subjected to miRNA and RNA sequencing. HFD and UC samples showed close similarities based on mRNA sequencing data. NG samples had a lower number of reads and different mRNA content compared to HFD or UC. For miRNA sequencing data, satisfactory miRNA counts were obtained by all methods, but miRNA contents differed slightly. This suggests that the isolation workflows enrich specific subpopulations of miRNA‐rich uEV preparation components. Our data shows that HFD,UC and the kit‐based method are suitable methods to isolate uEV for miRNA‐seq. However, only HFD and UC were suitable for mRNA‐seq in our settings. |
| doi_str_mv | 10.1002/jev2.12038 |
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However, comprehensive studies characterizing uEV isolation methods with patient samples are rare. Here we compared performance of three established uEV isolation workflows for their subsequent use in transcriptomics analysis for biomarker discovery in diabetic kidney disease. We collected urine samples from individuals with type 1 diabetes with macroalbuminuria and healthy controls. We isolated uEV by Hydrostatic Filtration Dialysis (HFD), ultracentrifugation (UC), and a commercial kit‐ based isolation method (NG), each with different established urine clearing steps. Purified EVs were analysed by electron microscopy, nanoparticle tracking analysis, and Western blotting. Isolated RNAs were subjected to miRNA and RNA sequencing. HFD and UC samples showed close similarities based on mRNA sequencing data. NG samples had a lower number of reads and different mRNA content compared to HFD or UC. For miRNA sequencing data, satisfactory miRNA counts were obtained by all methods, but miRNA contents differed slightly. This suggests that the isolation workflows enrich specific subpopulations of miRNA‐rich uEV preparation components. Our data shows that HFD,UC and the kit‐based method are suitable methods to isolate uEV for miRNA‐seq. However, only HFD and UC were suitable for mRNA‐seq in our settings.</description><identifier>ISSN: 2001-3078</identifier><identifier>EISSN: 2001-3078</identifier><identifier>DOI: 10.1002/jev2.12038</identifier><identifier>PMID: 33437407</identifier><language>eng</language><publisher>United States: John Wiley & Sons, Inc</publisher><subject>Adult ; Aged ; Biomarkers ; Biomarkers - urine ; Biopsy ; Case-Control Studies ; Cellulose acetate ; Creatinine ; Diabetes ; Diabetes mellitus (insulin dependent) ; Diabetes Mellitus, Type 1 - complications ; diabetic kidney disease ; Diabetic Nephropathies - diagnosis ; Diabetic Nephropathies - etiology ; Diabetic Nephropathies - metabolism ; Diabetic Nephropathies - urine ; Dialysis ; Electron microscopy ; exosomes ; Extracellular vesicles ; Extracellular Vesicles - genetics ; Extracellular Vesicles - metabolism ; Female ; Follow-Up Studies ; Gene Expression Regulation ; Hemodialysis ; Humans ; isolation ; Kidney diseases ; Lipids ; Male ; Methods ; MicroRNAs - genetics ; Middle Aged ; miRNA ; miRNA sequencing ; mRNA sequencing ; Nanoparticles ; Prognosis ; Proteins ; Transcriptome ; Transcriptomics ; Ultracentrifugation ; urinary extracellular vesicles ; Urine ; Western blotting</subject><ispartof>Journal of extracellular vesicles, 2020-12, Vol.10 (2), p.e12038-n/a</ispartof><rights>2020 The Authors. published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles</rights><rights>2020 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.</rights><rights>2020. This work is published under http://creativecommons.org/licenses/by-nc/4.0/ (the "License"). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5148-a238d63fe5ecbc6db7c524b4b930e7001381f551a0a87d32c017b02a52845c1c3</citedby><cites>FETCH-LOGICAL-c5148-a238d63fe5ecbc6db7c524b4b930e7001381f551a0a87d32c017b02a52845c1c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789228/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789228/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,1417,11562,27924,27925,45574,45575,46052,46476,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33437407$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Barreiro, Karina</creatorcontrib><creatorcontrib>Dwivedi, Om Prakash</creatorcontrib><creatorcontrib>Leparc, German</creatorcontrib><creatorcontrib>Rolser, Marcel</creatorcontrib><creatorcontrib>Delic, Denis</creatorcontrib><creatorcontrib>Forsblom, Carol</creatorcontrib><creatorcontrib>Groop, Per‐Henrik</creatorcontrib><creatorcontrib>Groop, Leif</creatorcontrib><creatorcontrib>Huber, Tobias B.</creatorcontrib><creatorcontrib>Puhka, Maija</creatorcontrib><creatorcontrib>Holthofer, Harry</creatorcontrib><title>Comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease</title><title>Journal of extracellular vesicles</title><addtitle>J Extracell Vesicles</addtitle><description>Urinary Extracellular Vesicles (uEV) have emerged as a source for biomarkers of kidney damage, holding potential to replace the conventional invasive techniques including kidney biopsy. However, comprehensive studies characterizing uEV isolation methods with patient samples are rare. Here we compared performance of three established uEV isolation workflows for their subsequent use in transcriptomics analysis for biomarker discovery in diabetic kidney disease. We collected urine samples from individuals with type 1 diabetes with macroalbuminuria and healthy controls. We isolated uEV by Hydrostatic Filtration Dialysis (HFD), ultracentrifugation (UC), and a commercial kit‐ based isolation method (NG), each with different established urine clearing steps. Purified EVs were analysed by electron microscopy, nanoparticle tracking analysis, and Western blotting. Isolated RNAs were subjected to miRNA and RNA sequencing. HFD and UC samples showed close similarities based on mRNA sequencing data. NG samples had a lower number of reads and different mRNA content compared to HFD or UC. For miRNA sequencing data, satisfactory miRNA counts were obtained by all methods, but miRNA contents differed slightly. This suggests that the isolation workflows enrich specific subpopulations of miRNA‐rich uEV preparation components. Our data shows that HFD,UC and the kit‐based method are suitable methods to isolate uEV for miRNA‐seq. However, only HFD and UC were suitable for mRNA‐seq in our settings.</description><subject>Adult</subject><subject>Aged</subject><subject>Biomarkers</subject><subject>Biomarkers - urine</subject><subject>Biopsy</subject><subject>Case-Control Studies</subject><subject>Cellulose acetate</subject><subject>Creatinine</subject><subject>Diabetes</subject><subject>Diabetes mellitus (insulin dependent)</subject><subject>Diabetes Mellitus, Type 1 - complications</subject><subject>diabetic kidney disease</subject><subject>Diabetic Nephropathies - diagnosis</subject><subject>Diabetic Nephropathies - etiology</subject><subject>Diabetic Nephropathies - metabolism</subject><subject>Diabetic Nephropathies - urine</subject><subject>Dialysis</subject><subject>Electron microscopy</subject><subject>exosomes</subject><subject>Extracellular vesicles</subject><subject>Extracellular Vesicles - genetics</subject><subject>Extracellular Vesicles - metabolism</subject><subject>Female</subject><subject>Follow-Up Studies</subject><subject>Gene Expression Regulation</subject><subject>Hemodialysis</subject><subject>Humans</subject><subject>isolation</subject><subject>Kidney diseases</subject><subject>Lipids</subject><subject>Male</subject><subject>Methods</subject><subject>MicroRNAs - genetics</subject><subject>Middle Aged</subject><subject>miRNA</subject><subject>miRNA sequencing</subject><subject>mRNA sequencing</subject><subject>Nanoparticles</subject><subject>Prognosis</subject><subject>Proteins</subject><subject>Transcriptome</subject><subject>Transcriptomics</subject><subject>Ultracentrifugation</subject><subject>urinary extracellular vesicles</subject><subject>Urine</subject><subject>Western blotting</subject><issn>2001-3078</issn><issn>2001-3078</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>24P</sourceid><sourceid>WIN</sourceid><sourceid>EIF</sourceid><recordid>eNp9kV9PFDEUxRuiEYK8-AFIE16MyWL_TGnnxcRsUCEkvgivTadzh-0y0y7tzOK--Nm96wJBH-xL25xfTs69h5B3nJ1yxsTHJazFKRdMmj1yIBjjM8m0efXivU-OSlkyPHXFlanfkH0pK6krpg_Ir3kaVi6HkiJNHZ1yiC5vKPwcs_PQ91PvMl1DCb4HilTvxoDoAOMitYV2KVMkY_E5rMY0BE-bkAaX7yDTDAVc9gsaIm2Da2BE-S60ETb4R63AW_K6c32Bo8f7kFx_Of8x_za7-v71Yv75auYVr8zMCWnaM9mBAt_4s7bRXomqqZpaMtA4qDS8U4o75oxupfCM64YJp4SplOdeHpJPO9_V1AzQeoiYurerHDDrxiYX7N9KDAt7m9ZWa1MLYdDg_aNBTvcTlNEOoWwX5CKkqVhRaa04rxlD9OQfdJmmHHE8K1ktcPW1UUh92FE-p1IydM9hOLPbZu22WfunWYSPX8Z_Rp96RIDvgIfQw-Y_Vvby_EbsTH8D_3uwsw</recordid><startdate>202012</startdate><enddate>202012</enddate><creator>Barreiro, Karina</creator><creator>Dwivedi, Om Prakash</creator><creator>Leparc, German</creator><creator>Rolser, Marcel</creator><creator>Delic, Denis</creator><creator>Forsblom, Carol</creator><creator>Groop, Per‐Henrik</creator><creator>Groop, Leif</creator><creator>Huber, Tobias B.</creator><creator>Puhka, Maija</creator><creator>Holthofer, Harry</creator><general>John Wiley & Sons, Inc</general><general>John Wiley and Sons Inc</general><scope>24P</scope><scope>WIN</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QP</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>202012</creationdate><title>Comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease</title><author>Barreiro, Karina ; Dwivedi, Om Prakash ; Leparc, German ; Rolser, Marcel ; Delic, Denis ; Forsblom, Carol ; Groop, Per‐Henrik ; Groop, Leif ; Huber, Tobias B. ; Puhka, Maija ; Holthofer, Harry</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5148-a238d63fe5ecbc6db7c524b4b930e7001381f551a0a87d32c017b02a52845c1c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Adult</topic><topic>Aged</topic><topic>Biomarkers</topic><topic>Biomarkers - urine</topic><topic>Biopsy</topic><topic>Case-Control Studies</topic><topic>Cellulose acetate</topic><topic>Creatinine</topic><topic>Diabetes</topic><topic>Diabetes mellitus (insulin dependent)</topic><topic>Diabetes Mellitus, Type 1 - complications</topic><topic>diabetic kidney disease</topic><topic>Diabetic Nephropathies - diagnosis</topic><topic>Diabetic Nephropathies - etiology</topic><topic>Diabetic Nephropathies - metabolism</topic><topic>Diabetic Nephropathies - urine</topic><topic>Dialysis</topic><topic>Electron microscopy</topic><topic>exosomes</topic><topic>Extracellular vesicles</topic><topic>Extracellular Vesicles - genetics</topic><topic>Extracellular Vesicles - metabolism</topic><topic>Female</topic><topic>Follow-Up Studies</topic><topic>Gene Expression Regulation</topic><topic>Hemodialysis</topic><topic>Humans</topic><topic>isolation</topic><topic>Kidney diseases</topic><topic>Lipids</topic><topic>Male</topic><topic>Methods</topic><topic>MicroRNAs - genetics</topic><topic>Middle Aged</topic><topic>miRNA</topic><topic>miRNA sequencing</topic><topic>mRNA sequencing</topic><topic>Nanoparticles</topic><topic>Prognosis</topic><topic>Proteins</topic><topic>Transcriptome</topic><topic>Transcriptomics</topic><topic>Ultracentrifugation</topic><topic>urinary extracellular vesicles</topic><topic>Urine</topic><topic>Western blotting</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Barreiro, Karina</creatorcontrib><creatorcontrib>Dwivedi, Om Prakash</creatorcontrib><creatorcontrib>Leparc, German</creatorcontrib><creatorcontrib>Rolser, Marcel</creatorcontrib><creatorcontrib>Delic, Denis</creatorcontrib><creatorcontrib>Forsblom, Carol</creatorcontrib><creatorcontrib>Groop, Per‐Henrik</creatorcontrib><creatorcontrib>Groop, Leif</creatorcontrib><creatorcontrib>Huber, Tobias B.</creatorcontrib><creatorcontrib>Puhka, Maija</creatorcontrib><creatorcontrib>Holthofer, Harry</creatorcontrib><collection>Wiley-Blackwell Open Access Titles</collection><collection>Wiley Free Content</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of extracellular vesicles</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Barreiro, Karina</au><au>Dwivedi, Om Prakash</au><au>Leparc, German</au><au>Rolser, Marcel</au><au>Delic, Denis</au><au>Forsblom, Carol</au><au>Groop, Per‐Henrik</au><au>Groop, Leif</au><au>Huber, Tobias B.</au><au>Puhka, Maija</au><au>Holthofer, Harry</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease</atitle><jtitle>Journal of extracellular vesicles</jtitle><addtitle>J Extracell Vesicles</addtitle><date>2020-12</date><risdate>2020</risdate><volume>10</volume><issue>2</issue><spage>e12038</spage><epage>n/a</epage><pages>e12038-n/a</pages><issn>2001-3078</issn><eissn>2001-3078</eissn><abstract>Urinary Extracellular Vesicles (uEV) have emerged as a source for biomarkers of kidney damage, holding potential to replace the conventional invasive techniques including kidney biopsy. However, comprehensive studies characterizing uEV isolation methods with patient samples are rare. Here we compared performance of three established uEV isolation workflows for their subsequent use in transcriptomics analysis for biomarker discovery in diabetic kidney disease. We collected urine samples from individuals with type 1 diabetes with macroalbuminuria and healthy controls. We isolated uEV by Hydrostatic Filtration Dialysis (HFD), ultracentrifugation (UC), and a commercial kit‐ based isolation method (NG), each with different established urine clearing steps. Purified EVs were analysed by electron microscopy, nanoparticle tracking analysis, and Western blotting. Isolated RNAs were subjected to miRNA and RNA sequencing. HFD and UC samples showed close similarities based on mRNA sequencing data. NG samples had a lower number of reads and different mRNA content compared to HFD or UC. For miRNA sequencing data, satisfactory miRNA counts were obtained by all methods, but miRNA contents differed slightly. This suggests that the isolation workflows enrich specific subpopulations of miRNA‐rich uEV preparation components. Our data shows that HFD,UC and the kit‐based method are suitable methods to isolate uEV for miRNA‐seq. However, only HFD and UC were suitable for mRNA‐seq in our settings.</abstract><cop>United States</cop><pub>John Wiley & Sons, Inc</pub><pmid>33437407</pmid><doi>10.1002/jev2.12038</doi><tpages>18</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Adult Aged Biomarkers Biomarkers - urine Biopsy Case-Control Studies Cellulose acetate Creatinine Diabetes Diabetes mellitus (insulin dependent) Diabetes Mellitus, Type 1 - complications diabetic kidney disease Diabetic Nephropathies - diagnosis Diabetic Nephropathies - etiology Diabetic Nephropathies - metabolism Diabetic Nephropathies - urine Dialysis Electron microscopy exosomes Extracellular vesicles Extracellular Vesicles - genetics Extracellular Vesicles - metabolism Female Follow-Up Studies Gene Expression Regulation Hemodialysis Humans isolation Kidney diseases Lipids Male Methods MicroRNAs - genetics Middle Aged miRNA miRNA sequencing mRNA sequencing Nanoparticles Prognosis Proteins Transcriptome Transcriptomics Ultracentrifugation urinary extracellular vesicles Urine Western blotting |
| title | Comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease |
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