Development of glomerulus-, tubule-, and collecting duct-specific mRNA assay in human urinary exosomes and microvesicles
Urinary exosomes and microvesicles (EMV) are promising biomarkers for renal diseases. Although the density of EMV is very low in urine, large quantity of urine can be easily obtained. In order to analyze urinary EMV mRNA, a unique filter device to adsorb urinary EMV from 10 mL urine was developed, w...
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| Veröffentlicht in: | PloS one 2014-10, Vol.9 (9), p.e109074-e109074 |
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| description | Urinary exosomes and microvesicles (EMV) are promising biomarkers for renal diseases. Although the density of EMV is very low in urine, large quantity of urine can be easily obtained. In order to analyze urinary EMV mRNA, a unique filter device to adsorb urinary EMV from 10 mL urine was developed, which is far more convenient than the standard ultracentrifugation protocol. The filter part of the device is detachable and aligned to a 96-well microplate format, therefore multiple samples can be processed simultaneously in a high throughput manner following the isolation step. For EMV mRNA quantification, the EMV on the filter is lysed directly by adding lysis buffer and transferred to an oligo(dT)-immobilized microplate for mRNA isolation followed by cDNA synthesis and real-time PCR. Under the optimized assay condition, our method provided comparable or even superior results to the standard ultracentrifugation method in terms of mRNA assay sensitivity, linearity, intra-assay reproducibility, and ease of use. The assay system was applied to quantification of kidney-specific mRNAs such as NPHN and PDCN (glomerular filtration), SLC12A1 (tubular absorption), UMOD and ALB (tubular secretion), and AQP2 (collecting duct water absorption). 12-hour urine samples were collected from four healthy subjects for two weeks, and day-to-day and individual-to-individual variations were investigated. Kidney-specific genes as well as control genes (GAPDH, ACTB, etc.) were successfully detected and confirmed their stable expressions through the two-week study period. In conclusion, this method is readily available to clinical studies of kidney diseases. |
| doi_str_mv | 10.1371/journal.pone.0109074 |
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Although the density of EMV is very low in urine, large quantity of urine can be easily obtained. In order to analyze urinary EMV mRNA, a unique filter device to adsorb urinary EMV from 10 mL urine was developed, which is far more convenient than the standard ultracentrifugation protocol. The filter part of the device is detachable and aligned to a 96-well microplate format, therefore multiple samples can be processed simultaneously in a high throughput manner following the isolation step. For EMV mRNA quantification, the EMV on the filter is lysed directly by adding lysis buffer and transferred to an oligo(dT)-immobilized microplate for mRNA isolation followed by cDNA synthesis and real-time PCR. Under the optimized assay condition, our method provided comparable or even superior results to the standard ultracentrifugation method in terms of mRNA assay sensitivity, linearity, intra-assay reproducibility, and ease of use. The assay system was applied to quantification of kidney-specific mRNAs such as NPHN and PDCN (glomerular filtration), SLC12A1 (tubular absorption), UMOD and ALB (tubular secretion), and AQP2 (collecting duct water absorption). 12-hour urine samples were collected from four healthy subjects for two weeks, and day-to-day and individual-to-individual variations were investigated. Kidney-specific genes as well as control genes (GAPDH, ACTB, etc.) were successfully detected and confirmed their stable expressions through the two-week study period. In conclusion, this method is readily available to clinical studies of kidney diseases.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0109074</identifier><identifier>PMID: 25275511</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Absorption ; Analysis ; Aquaporin 2 ; Aquaporins ; Assaying ; Bioindicators ; Biological Assay - methods ; Biology and Life Sciences ; Biomarkers ; Blood ; Collecting duct ; Exosomes ; Exosomes - metabolism ; Exosomes - ultrastructure ; Genes ; Glomerulus ; Glyceraldehyde-3-phosphate dehydrogenase ; Humans ; Hydrogen-Ion Concentration ; Kidney diseases ; Kidney Glomerulus - metabolism ; Kidney Tubules, Collecting - metabolism ; Kidneys ; Linearity ; Lysis ; Medicine and Health Sciences ; Messenger RNA ; Methods ; MicroRNAs ; mRNA ; Organ Specificity ; Plasma ; Proteins ; Reference Standards ; Reproducibility ; Research and analysis methods ; RNA, Messenger - genetics ; RNA, Messenger - urine ; Salts - chemistry ; Secretion ; Ultracentrifugation ; Urine ; Water absorption</subject><ispartof>PloS one, 2014-10, Vol.9 (9), p.e109074-e109074</ispartof><rights>COPYRIGHT 2014 Public Library of Science</rights><rights>2014 Murakami et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2014 Murakami et al 2014 Murakami et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c762t-ef3e4b1a8065218b009bb3e70eb5bbd37decd8ff383943dedf1c7f36d8dbbe253</citedby><cites>FETCH-LOGICAL-c762t-ef3e4b1a8065218b009bb3e70eb5bbd37decd8ff383943dedf1c7f36d8dbbe253</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/PMC4183527/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4183527/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79343,79344</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25275511$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Feraille, Eric</contributor><creatorcontrib>Murakami, Taku</creatorcontrib><creatorcontrib>Oakes, Melanie</creatorcontrib><creatorcontrib>Ogura, Mieko</creatorcontrib><creatorcontrib>Tovar, Vivian</creatorcontrib><creatorcontrib>Yamamoto, Cindy</creatorcontrib><creatorcontrib>Mitsuhashi, Masato</creatorcontrib><title>Development of glomerulus-, tubule-, and collecting duct-specific mRNA assay in human urinary exosomes and microvesicles</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Urinary exosomes and microvesicles (EMV) are promising biomarkers for renal diseases. Although the density of EMV is very low in urine, large quantity of urine can be easily obtained. In order to analyze urinary EMV mRNA, a unique filter device to adsorb urinary EMV from 10 mL urine was developed, which is far more convenient than the standard ultracentrifugation protocol. The filter part of the device is detachable and aligned to a 96-well microplate format, therefore multiple samples can be processed simultaneously in a high throughput manner following the isolation step. For EMV mRNA quantification, the EMV on the filter is lysed directly by adding lysis buffer and transferred to an oligo(dT)-immobilized microplate for mRNA isolation followed by cDNA synthesis and real-time PCR. Under the optimized assay condition, our method provided comparable or even superior results to the standard ultracentrifugation method in terms of mRNA assay sensitivity, linearity, intra-assay reproducibility, and ease of use. The assay system was applied to quantification of kidney-specific mRNAs such as NPHN and PDCN (glomerular filtration), SLC12A1 (tubular absorption), UMOD and ALB (tubular secretion), and AQP2 (collecting duct water absorption). 12-hour urine samples were collected from four healthy subjects for two weeks, and day-to-day and individual-to-individual variations were investigated. Kidney-specific genes as well as control genes (GAPDH, ACTB, etc.) were successfully detected and confirmed their stable expressions through the two-week study period. In conclusion, this method is readily available to clinical studies of kidney diseases.</description><subject>Absorption</subject><subject>Analysis</subject><subject>Aquaporin 2</subject><subject>Aquaporins</subject><subject>Assaying</subject><subject>Bioindicators</subject><subject>Biological Assay - methods</subject><subject>Biology and Life Sciences</subject><subject>Biomarkers</subject><subject>Blood</subject><subject>Collecting duct</subject><subject>Exosomes</subject><subject>Exosomes - metabolism</subject><subject>Exosomes - ultrastructure</subject><subject>Genes</subject><subject>Glomerulus</subject><subject>Glyceraldehyde-3-phosphate dehydrogenase</subject><subject>Humans</subject><subject>Hydrogen-Ion Concentration</subject><subject>Kidney diseases</subject><subject>Kidney Glomerulus - metabolism</subject><subject>Kidney Tubules, Collecting - metabolism</subject><subject>Kidneys</subject><subject>Linearity</subject><subject>Lysis</subject><subject>Medicine and Health Sciences</subject><subject>Messenger RNA</subject><subject>Methods</subject><subject>MicroRNAs</subject><subject>mRNA</subject><subject>Organ Specificity</subject><subject>Plasma</subject><subject>Proteins</subject><subject>Reference Standards</subject><subject>Reproducibility</subject><subject>Research and analysis methods</subject><subject>RNA, Messenger - genetics</subject><subject>RNA, Messenger - urine</subject><subject>Salts - chemistry</subject><subject>Secretion</subject><subject>Ultracentrifugation</subject><subject>Urine</subject><subject>Water absorption</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNk11r2zAUhs3YWLtu_2BshsHYYMkky5aUm0HovgJlhe7jVsjSUaIgW6llhfTfT27ckoxeDF9YSM95z9F7dLLsJUZTTBj-uPaxa6WbbnwLU4TRDLHyUXaKZ6SY0AKRxwfrk-xZCGuEKsIpfZqdFFXBqgrj02z3Gbbg_KaBts-9yZfON9BFF8PkQ97HOjpIC9nqXHnnQPW2XeY6qn4SNqCssSpvrn7McxmCvMltm69iI9s8draV3U0OOx-SYLhVaKzq_BaCVQ7C8-yJkS7Ai_F_lv3--uXX-ffJxeW3xfn8YqIYLfoJGAJljSVHtCowrxGa1TUBhqCu6loTpkFpbgzhZFYSDdpgxQyhmuu6hqIiZ9nrve7G-SBG04LAFWWo5AVliVjsCe3lWmw626TKhZdW3G74bilk1w9FC4q0plJyjIqqNBhmVBW8TAYb4DVhkLQ-jdli3YBWydVOuiPR45PWrsTSb0WJOUlNSQLvRoHOX0cIvWhsUOCcbMHHoW5OU_YCDeibf9CHbzdSS5kuYFvjU141iIp5iSljjLPBpekDVPo0pK6lF2Zs2j8KeH8UkJgedv1SxhDE4ufV_7OXf47ZtwfsCqTrV8G72FvfhmOw3IPpTYXQgbk3GSMxDMidG2IYEDEOSAp7ddig-6C7iSB_Aea6DRc</recordid><startdate>20141002</startdate><enddate>20141002</enddate><creator>Murakami, Taku</creator><creator>Oakes, Melanie</creator><creator>Ogura, Mieko</creator><creator>Tovar, Vivian</creator><creator>Yamamoto, Cindy</creator><creator>Mitsuhashi, Masato</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><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>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20141002</creationdate><title>Development of glomerulus-, tubule-, and collecting duct-specific mRNA assay in human urinary exosomes and microvesicles</title><author>Murakami, Taku ; Oakes, Melanie ; Ogura, Mieko ; Tovar, Vivian ; Yamamoto, Cindy ; Mitsuhashi, Masato</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c762t-ef3e4b1a8065218b009bb3e70eb5bbd37decd8ff383943dedf1c7f36d8dbbe253</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Absorption</topic><topic>Analysis</topic><topic>Aquaporin 2</topic><topic>Aquaporins</topic><topic>Assaying</topic><topic>Bioindicators</topic><topic>Biological Assay - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Murakami, Taku</au><au>Oakes, Melanie</au><au>Ogura, Mieko</au><au>Tovar, Vivian</au><au>Yamamoto, Cindy</au><au>Mitsuhashi, Masato</au><au>Feraille, Eric</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of glomerulus-, tubule-, and collecting duct-specific mRNA assay in human urinary exosomes and microvesicles</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2014-10-02</date><risdate>2014</risdate><volume>9</volume><issue>9</issue><spage>e109074</spage><epage>e109074</epage><pages>e109074-e109074</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Urinary exosomes and microvesicles (EMV) are promising biomarkers for renal diseases. Although the density of EMV is very low in urine, large quantity of urine can be easily obtained. In order to analyze urinary EMV mRNA, a unique filter device to adsorb urinary EMV from 10 mL urine was developed, which is far more convenient than the standard ultracentrifugation protocol. The filter part of the device is detachable and aligned to a 96-well microplate format, therefore multiple samples can be processed simultaneously in a high throughput manner following the isolation step. For EMV mRNA quantification, the EMV on the filter is lysed directly by adding lysis buffer and transferred to an oligo(dT)-immobilized microplate for mRNA isolation followed by cDNA synthesis and real-time PCR. Under the optimized assay condition, our method provided comparable or even superior results to the standard ultracentrifugation method in terms of mRNA assay sensitivity, linearity, intra-assay reproducibility, and ease of use. The assay system was applied to quantification of kidney-specific mRNAs such as NPHN and PDCN (glomerular filtration), SLC12A1 (tubular absorption), UMOD and ALB (tubular secretion), and AQP2 (collecting duct water absorption). 12-hour urine samples were collected from four healthy subjects for two weeks, and day-to-day and individual-to-individual variations were investigated. Kidney-specific genes as well as control genes (GAPDH, ACTB, etc.) were successfully detected and confirmed their stable expressions through the two-week study period. In conclusion, this method is readily available to clinical studies of kidney diseases.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>25275511</pmid><doi>10.1371/journal.pone.0109074</doi><oa>free_for_read</oa></addata></record> |
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| subjects | Absorption Analysis Aquaporin 2 Aquaporins Assaying Bioindicators Biological Assay - methods Biology and Life Sciences Biomarkers Blood Collecting duct Exosomes Exosomes - metabolism Exosomes - ultrastructure Genes Glomerulus Glyceraldehyde-3-phosphate dehydrogenase Humans Hydrogen-Ion Concentration Kidney diseases Kidney Glomerulus - metabolism Kidney Tubules, Collecting - metabolism Kidneys Linearity Lysis Medicine and Health Sciences Messenger RNA Methods MicroRNAs mRNA Organ Specificity Plasma Proteins Reference Standards Reproducibility Research and analysis methods RNA, Messenger - genetics RNA, Messenger - urine Salts - chemistry Secretion Ultracentrifugation Urine Water absorption |
| title | Development of glomerulus-, tubule-, and collecting duct-specific mRNA assay in human urinary exosomes and microvesicles |
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