{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T13:24:42Z","timestamp":1787318682420,"version":"build-2736575974"},"reference-count":38,"publisher":"Public Library of Science (PLoS)","issue":"3","license":[{"start":{"date-parts":[[2026,3,13]],"date-time":"2026-03-13T00:00:00Z","timestamp":1773360000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003407","name":"Ministero dell\u2019Istruzione, dell\u2019Universit\u00e0 e della Ricerca","doi-asserted-by":"publisher","award":["PE0000006"],"award-info":[{"award-number":["PE0000006"]}],"id":[{"id":"10.13039\/501100003407","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100007074","name":"Fondazione CON IL SUD","doi-asserted-by":"publisher","award":["2018-PDR-00351"],"award-info":[{"award-number":["2018-PDR-00351"]}],"id":[{"id":"10.13039\/501100007074","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100005362","name":"Universita degli Studi di Bari Aldo Moro","doi-asserted-by":"publisher","award":["0245439"],"award-info":[{"award-number":["0245439"]}],"id":[{"id":"10.13039\/501100005362","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    Patient-derived cell models of dystrophic myogenesis and differentiation are valuable preclinical tools for early and mutation-based assessment of candidate therapeutic approaches. Quantitative measurement of gene expression within such models plays a key role in these studies, but normalisation of RT-qPCR data requires a panel of validated stably expressed reference genes. This study aims to identify stable reference genes for RT-qPCR assays in three human derived muscle immortalized cell lines: one healthy WT (from a 16-year-old donor), and two dystrophic lines, DMD1 (from a 11-year-old patient carrying a stop codon mutation on exon 59) and DMD2, from a 14-year-old patient carrying an exon 48\u201350 deletion. We screened a pool of 14 candidate genes (\n                    <jats:italic>ACTB, HPRT1, RPL13A, RPS18, GAPDH, ALAS1, UBC, YWHAZ, IPO8, PSMC4, HSP90AB1, NONO,<\/jats:italic>\n                    <jats:italic>CSNK2A2, AP3D1<\/jats:italic>\n                    ), investigating stability of expression from proliferation through to 11 days of myogenic differentiation. Data were analysed using four complementary approaches (Bestkeeper, geNorm, Normfinder and DeltaCt) to determine the most appropriate references both within and between cell lines. Our study shows that\n                    <jats:italic>RPS18, UBC, YWHAZ<\/jats:italic>\n                    scored highly across all comparisons, and we therefore suggest that these three genes represent an appropriate reference panel for these human myogenic cell lines, regardless of genotype or differentiation stage.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0344973","type":"journal-article","created":{"date-parts":[[2026,3,13]],"date-time":"2026-03-13T17:24:05Z","timestamp":1773422645000},"page":"e0344973","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Best reference genes for unbiased normalized transcript expression in normal and dystrophic human cell models of myogenesis"],"prefix":"10.1371","volume":"21","author":[{"given":"Raffaella","family":"Quarta","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1078-8490","authenticated-orcid":true,"given":"Brigida","family":"Boccanegra","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Enrica","family":"Cristiano","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alberto","family":"Ladisa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elena","family":"Conte","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jessica","family":"Ohana","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vincent","family":"Mouly","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Annamaria","family":"De 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types and paradoxical cases that confirm the reading-frame rule","volume":"34","author":"A Aartsma-Rus","year":"2006","journal-title":"Muscle Nerve"},{"issue":"3","key":"pone.0344973.ref003","first-page":"243","article-title":"Muscle stem cells in developmental and regenerative myogenesis","volume":"13","author":"J-S Kang","year":"2010","journal-title":"Curr Opin Clin Nutr Metab Care"},{"key":"pone.0344973.ref004","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.semcdb.2017.11.010","article-title":"The myogenic regulatory factors, determinants of muscle development, cell identity and regeneration","volume":"72","author":"JM Hern\u00e1ndez-Hern\u00e1ndez","year":"2017","journal-title":"Semin Cell Dev Biol"},{"key":"pone.0344973.ref005","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1186\/1471-2407-10-240","article-title":"Identification of valid reference genes for gene expression studies of human stomach cancer by reverse transcription-qPCR","volume":"10","author":"H-W Rho","year":"2010","journal-title":"BMC Cancer"},{"issue":"11","key":"pone.0344973.ref006","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0310714","article-title":"Determination of qPCR reference genes suitable for normalizing gene expression in a novel model of Duchenne muscular dystrophy, the D2-mdx mouse","volume":"19","author":"B Boccanegra","year":"2024","journal-title":"PLoS One"},{"key":"pone.0344973.ref007","unstructured":"Glare EM, Divjak M, Bailey MJ, Walters EH. \u03b2-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels. 2023"},{"issue":"14","key":"pone.0344973.ref008","doi-asserted-by":"crossref","first-page":"5815","DOI":"10.7314\/APJCP.2014.15.14.5815","article-title":"Optimization of reference genes for normalization of the quantitative polymerase chain reaction in tissue samples of gastric cancer","volume":"15","author":"L-M 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Notes"},{"issue":"19","key":"pone.0344973.ref032","doi-asserted-by":"crossref","first-page":"16673","DOI":"10.1074\/jbc.M200501200","article-title":"Ubiquitin (UbC) expression in muscle cells is increased by glucocorticoids through a mechanism involving Sp1 and MEK1","volume":"277","author":"AC Marinovic","year":"2002","journal-title":"J Biol Chem"},{"key":"pone.0344973.ref033","article-title":"Comparison of reference gene expression stability in mouse skeletal muscle via five algorithms","volume":"10","author":"J Ma","year":"2022","journal-title":"PeerJ"},{"issue":"3","key":"pone.0344973.ref034","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1016\/j.devcel.2013.12.020","article-title":"Gene regulatory networks and transcriptional mechanisms that control myogenesis","volume":"28","author":"M Buckingham","year":"2014","journal-title":"Dev Cell"},{"issue":"3","key":"pone.0344973.ref035","doi-asserted-by":"crossref","first-page":"383","DOI":"10.1016\/j.coph.2012.02.001","article-title":"MicroRNAs regulate and provide robustness to the myogenic transcriptional network","volume":"12","author":"J Gagan","year":"2012","journal-title":"Curr Opin Pharmacol"},{"issue":"6","key":"pone.0344973.ref036","doi-asserted-by":"crossref","first-page":"2054","DOI":"10.1172\/JCI62656","article-title":"microRNA-206 promotes skeletal muscle regeneration and delays progression of Duchenne muscular dystrophy in mice","volume":"122","author":"N Liu","year":"2012","journal-title":"J Clin Invest"},{"issue":"5","key":"pone.0344973.ref037","first-page":"4377","article-title":"Identification of suitable reference genes for gene expression studies in rat skeletal muscle following sciatic nerve crush injury","volume":"19","author":"X Wang","year":"2019","journal-title":"Mol Med Rep"},{"issue":"2","key":"pone.0344973.ref038","doi-asserted-by":"crossref","first-page":"177","DOI":"10.3233\/JND-170267","article-title":"Determination of qPCR Reference Genes Suitable for Normalizing Gene Expression in a Canine Model of Duchenne Muscular Dystrophy","volume":"5","author":"JCW Hildyard","year":"2018","journal-title":"J Neuromuscul Dis"}],"container-title":["PLOS 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