Selection of reference miRNAs for RT-qPCR assays in endometriosis menstrual blood-derived mesenchymal stem cells
Choosing appropriate reference genes or internal controls to normalize RT-qPCR data is mandatory for the interexperimental reproducibility of gene expression data obtained by RT-qPCR in most studies, including those on endometriosis. Particularly for miRNAs, the choice for reference genes is challen...
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| creator | Hacimoto, Sabrina Yukari Santos Cressoni, Ana Clara Lagazzi Silva, Lilian Eslaine Costa Mendes da Padovan, Cristiana Carolina Ferriani, Rui Alberto Rosa-E-Silva, Júlio César Meola, Juliana |
| description | Choosing appropriate reference genes or internal controls to normalize RT-qPCR data is mandatory for the interexperimental reproducibility of gene expression data obtained by RT-qPCR in most studies, including those on endometriosis. Particularly for miRNAs, the choice for reference genes is challenging because of their physicochemical and biological characteristics. Moreover, the retrograde menstruation theory, mesenchymal stem cells in menstrual blood (MenSCs), and changes in post-transcriptional regulatory processes through miRNAs have gained prominence in the scientific community as important players in endometriosis. Therefore, we originally explored the stability of 10 miRNAs expressions as internal control candidates in conditions involving the two-dimensional culture of MenSCs from healthy women and patients with endometriosis. Here, we applied multiple algorithms (geNorm, NormFinder, Bestkeeper, and delta Ct) to screen reference genes and assessed the comprehensive stability classification of miRNAs using RefFinder. Pairwise variation calculated using geNorm identified three miRNAs as a sufficient number of reference genes for accurate normalization. MiR-191-5p, miR-24-3p, and miR-103a-3p were the best combination for suitable gene expression normalization. This study will benefit similar research, but is also attractive for regenerative medicine and clinics that use MenSCs, miRNA expression, and RT-qPCR. |
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Particularly for miRNAs, the choice for reference genes is challenging because of their physicochemical and biological characteristics. Moreover, the retrograde menstruation theory, mesenchymal stem cells in menstrual blood (MenSCs), and changes in post-transcriptional regulatory processes through miRNAs have gained prominence in the scientific community as important players in endometriosis. Therefore, we originally explored the stability of 10 miRNAs expressions as internal control candidates in conditions involving the two-dimensional culture of MenSCs from healthy women and patients with endometriosis. Here, we applied multiple algorithms (geNorm, NormFinder, Bestkeeper, and delta Ct) to screen reference genes and assessed the comprehensive stability classification of miRNAs using RefFinder. Pairwise variation calculated using geNorm identified three miRNAs as a sufficient number of reference genes for accurate normalization. MiR-191-5p, miR-24-3p, and miR-103a-3p were the best combination for suitable gene expression normalization. This study will benefit similar research, but is also attractive for regenerative medicine and clinics that use MenSCs, miRNA expression, and RT-qPCR.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0306657</identifier><identifier>PMID: 39078824</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Adult ; Algorithms ; Biology and life sciences ; Blood ; Cell culture ; Chromosome 3 ; Chromosome 5 ; Computer and Information Sciences ; Control stability ; Dimensional stability ; Disease ; Endometriosis ; Endometriosis - genetics ; Engineering and Technology ; Ethics ; Ethylenediaminetetraacetic acid ; Female ; Gene expression ; Gene Expression Profiling - methods ; Genes ; Humans ; Instrument industry ; Medical research ; Medicine and Health Sciences ; Menstruation ; Menstruation - genetics ; Mesenchymal stem cells ; Mesenchymal Stem Cells - metabolism ; MicroRNA ; MicroRNAs ; MicroRNAs - genetics ; miRNA ; Molecular biology ; Physical Sciences ; Post-transcription ; Real-Time Polymerase Chain Reaction - methods ; Real-Time Polymerase Chain Reaction - standards ; Reference Standards ; Regenerative medicine ; Reproducibility of Results ; Research and Analysis Methods ; Stem cells ; Womens health</subject><ispartof>PloS one, 2024-07, Vol.19 (7), p.e0306657</ispartof><rights>Copyright: © 2024 Hacimoto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</rights><rights>COPYRIGHT 2024 Public Library of Science</rights><rights>2024 Hacimoto 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>2024 Hacimoto et al 2024 Hacimoto et al</rights><rights>2024 Hacimoto et al. 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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><cites>FETCH-LOGICAL-c572t-21cbe21762f8dbf70fccb8b9fb6ec4f98dd6701f87bd1ba684d0d3eb18f1a1d53</cites><orcidid>0000-0003-2514-4011</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC11288454/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC11288454/$$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/39078824$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hacimoto, Sabrina Yukari Santos</creatorcontrib><creatorcontrib>Cressoni, Ana Clara Lagazzi</creatorcontrib><creatorcontrib>Silva, Lilian Eslaine Costa Mendes da</creatorcontrib><creatorcontrib>Padovan, Cristiana Carolina</creatorcontrib><creatorcontrib>Ferriani, Rui Alberto</creatorcontrib><creatorcontrib>Rosa-E-Silva, Júlio César</creatorcontrib><creatorcontrib>Meola, Juliana</creatorcontrib><title>Selection of reference miRNAs for RT-qPCR assays in endometriosis menstrual blood-derived mesenchymal stem cells</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Choosing appropriate reference genes or internal controls to normalize RT-qPCR data is mandatory for the interexperimental reproducibility of gene expression data obtained by RT-qPCR in most studies, including those on endometriosis. 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MiR-191-5p, miR-24-3p, and miR-103a-3p were the best combination for suitable gene expression normalization. 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Particularly for miRNAs, the choice for reference genes is challenging because of their physicochemical and biological characteristics. Moreover, the retrograde menstruation theory, mesenchymal stem cells in menstrual blood (MenSCs), and changes in post-transcriptional regulatory processes through miRNAs have gained prominence in the scientific community as important players in endometriosis. Therefore, we originally explored the stability of 10 miRNAs expressions as internal control candidates in conditions involving the two-dimensional culture of MenSCs from healthy women and patients with endometriosis. Here, we applied multiple algorithms (geNorm, NormFinder, Bestkeeper, and delta Ct) to screen reference genes and assessed the comprehensive stability classification of miRNAs using RefFinder. Pairwise variation calculated using geNorm identified three miRNAs as a sufficient number of reference genes for accurate normalization. MiR-191-5p, miR-24-3p, and miR-103a-3p were the best combination for suitable gene expression normalization. This study will benefit similar research, but is also attractive for regenerative medicine and clinics that use MenSCs, miRNA expression, and RT-qPCR.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>39078824</pmid><doi>10.1371/journal.pone.0306657</doi><tpages>e0306657</tpages><orcidid>https://orcid.org/0000-0003-2514-4011</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Adult Algorithms Biology and life sciences Blood Cell culture Chromosome 3 Chromosome 5 Computer and Information Sciences Control stability Dimensional stability Disease Endometriosis Endometriosis - genetics Engineering and Technology Ethics Ethylenediaminetetraacetic acid Female Gene expression Gene Expression Profiling - methods Genes Humans Instrument industry Medical research Medicine and Health Sciences Menstruation Menstruation - genetics Mesenchymal stem cells Mesenchymal Stem Cells - metabolism MicroRNA MicroRNAs MicroRNAs - genetics miRNA Molecular biology Physical Sciences Post-transcription Real-Time Polymerase Chain Reaction - methods Real-Time Polymerase Chain Reaction - standards Reference Standards Regenerative medicine Reproducibility of Results Research and Analysis Methods Stem cells Womens health |
| title | Selection of reference miRNAs for RT-qPCR assays in endometriosis menstrual blood-derived mesenchymal stem cells |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-04T01%3A41%3A10IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Selection%20of%20reference%20miRNAs%20for%20RT-qPCR%20assays%20in%20endometriosis%20menstrual%20blood-derived%20mesenchymal%20stem%20cells&rft.jtitle=PloS%20one&rft.au=Hacimoto,%20Sabrina%20Yukari%20Santos&rft.date=2024-07-30&rft.volume=19&rft.issue=7&rft.spage=e0306657&rft.pages=e0306657-&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0306657&rft_dat=%3Cgale_plos_%3EA803192802%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=3086285300&rft_id=info:pmid/39078824&rft_galeid=A803192802&rft_doaj_id=oai_doaj_org_article_69472de13ccf4fea8a16e6062a7de126&rfr_iscdi=true |