{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T12:53:38Z","timestamp":1787316818760,"version":"build-2736575974"},"reference-count":32,"publisher":"Public Library of Science (PLoS)","issue":"11","license":[{"start":{"date-parts":[[2024,11,13]],"date-time":"2024-11-13T00:00:00Z","timestamp":1731456000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"PNRR - AGE-IT","award":["PE_0000015"],"award-info":[{"award-number":["PE_0000015"]}]},{"name":"Of Mice and Measures OMAM - Charley's Fund"},{"name":"PRIN MUR","award":["2020 Prot. 2020ELYA32"],"award-info":[{"award-number":["2020 Prot. 2020ELYA32"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"\n Duchenne muscular dystrophy (DMD) is a X-linked neuromuscular disorder arising from mutations in the dystrophin gene, leading to a progressive muscle wasting and disability. Currently there is no universal therapy, and there is thus a strong interest in preclinical studies for finding novel treatments. The most widely used and characterized mouse model for DMD is the C57BL\/10ScSn-\n Dmd<\/jats:italic>\n \n mdx<\/jats:italic>\n <\/jats:sup>\n \/<\/jats:italic>\n J (BL10-\n mdx<\/jats:italic>\n ), but this model exhibits mild pathology and does not replicate key features of human disease. The D2.B10-\n Dmd<\/jats:italic>\n \n mdx<\/jats:italic>\n <\/jats:sup>\n \/J (D2-\n mdx<\/jats:italic>\n ) mouse is a more recent model which seems to better mimics the complex human DMD phenotype. However, the D2-\n mdx<\/jats:italic>\n mouse remains less extensively characterised than its BL10-\n mdx<\/jats:italic>\n counterpart. Quantitative PCR analysis of gene expression is an important tool to monitor disease progression and evaluate therapeutic efficacy, but measurements must be normalised to stably expressed reference genes, which should ideally be determined and validated empirically. We examined gene expression in the gastrocnemius (GC), diaphragm (DIA) and heart in the D2-\n md<\/jats:italic>\n x mouse, the BL10-\n mdx<\/jats:italic>\n mouse, and appropriate strain-matched wild-type controls (D2-wt and BL10-wt), from 4 to 52 weeks of age, using a large panel of candidate references (\n ACTB<\/jats:italic>\n ,\n AP3D1<\/jats:italic>\n ,\n CSNK2A2<\/jats:italic>\n ,\n GAPDH<\/jats:italic>\n ,\n HPRT1<\/jats:italic>\n ,\n PAK1IP1<\/jats:italic>\n ,\n RPL13A<\/jats:italic>\n ,\n SDHA<\/jats:italic>\n , and in the heart, also\n HTATSF1<\/jats:italic>\n and\n HMBS<\/jats:italic>\n ). Data was analyzed using GeNorm, Bestkeeper, deltaCt and Normfinder algorithms to identify stable references under multiple possible scenarios. We show that\n CSNK2A2<\/jats:italic>\n ,\n AP3D1<\/jats:italic>\n and\n ACTB<\/jats:italic>\n represent strong universal reference genes in both GC and DIA, regardless of age, muscle type, strain and genotype, while\n HTATSF1<\/jats:italic>\n and\n SDHA<\/jats:italic>\n are optimal for the heart.\n GAPDH<\/jats:italic>\n ,\n HPRT1<\/jats:italic>\n and\n RPL13A<\/jats:italic>\n were conversely revealed to be poor references, showing tissue-, age- or disease-specific changes in expression. Our results illustrate the importance of determining appropriate reference genes for specific comparative scenarios, but also reconfirm that universal panels can nevertheless be identified for normalising gene expression studies in even complex pathological states.\n <\/jats:p>","DOI":"10.1371\/journal.pone.0310714","type":"journal-article","created":{"date-parts":[[2024,11,13]],"date-time":"2024-11-13T13:24:43Z","timestamp":1731504283000},"page":"e0310714","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":5,"title":["Determination of qPCR reference genes suitable for normalizing gene expression in a novel model of Duchenne muscular dystrophy, the D2-mdx mouse"],"prefix":"10.1371","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1078-8490","authenticated-orcid":true,"given":"Brigida","family":"Boccanegra","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roberta","family":"Lenti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6339-2222","authenticated-orcid":true,"given":"Paola","family":"Mantuano","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elena","family":"Conte","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lisamaura","family":"Tulimiero","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard J.","family":"Piercy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1882-3502","authenticated-orcid":true,"given":"Ornella","family":"Cappellari","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2283-2118","authenticated-orcid":true,"given":"John C. W.","family":"Hildyard","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Annamaria","family":"De Luca","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2024,11,13]]},"reference":[{"issue":"1","key":"pone.0310714.ref001","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1038\/s41572-021-00248-3","article-title":"Duchenne muscular dystrophy","volume":"7","author":"D Duan","year":"2021","journal-title":"Nat Rev Dis Primers"},{"issue":"7","key":"pone.0310714.ref002","doi-asserted-by":"crossref","DOI":"10.3390\/cells9071659","article-title":"\u2018The Social Network\u2019 and Muscular Dystrophies: The Lesson Learnt about the Niche Environment as a Target for Therapeutic Strategies","volume":"9","author":"O Cappellari","year":"2020","journal-title":"Cells"},{"issue":"3","key":"pone.0310714.ref003","doi-asserted-by":"crossref","first-page":"482","DOI":"10.1093\/hmg\/ddn376","article-title":"Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy","volume":"18","author":"SA Villalta","year":"2009","journal-title":"Hum Mol Genet"},{"issue":"1","key":"pone.0310714.ref004","article-title":"The immune system in Duchenne muscular dystrophy: Friend or foe","volume":"3","author":"SA Villalta","year":"2015","journal-title":"Rare Dis"},{"issue":"3","key":"pone.0310714.ref005","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1002\/humu.20918","article-title":"Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations","volume":"30","author":"A Aartsma-Rus","year":"2009","journal-title":"Hum Mutat"},{"issue":"10","key":"pone.0310714.ref006","doi-asserted-by":"crossref","first-page":"1536","DOI":"10.3390\/biom13101536","article-title":"Pharmacotherapeutic Approaches to Treatment of Muscular Dystrophies","volume":"13","author":"A Rawls","year":"2023","journal-title":"Biomolecules"},{"key":"pone.0310714.ref007","doi-asserted-by":"crossref","first-page":"238","DOI":"10.12688\/wellcomeopenres.18251.1","article-title":"The skeletal muscle phenotype of the DE50-MD dog model of Duchenne muscular dystrophy","volume":"7","author":"JCW Hildyard","year":"2022","journal-title":"Wellcome Open Res"},{"issue":"12","key":"pone.0310714.ref008","doi-asserted-by":"crossref","first-page":"dmm049285","DOI":"10.1242\/dmm.049285","article-title":"A scalable, clinically severe pig model for Duchenne muscular dystrophy","volume":"14","author":"M Stirm","year":"2021","journal-title":"Disease Models & Mechanisms"},{"issue":"1","key":"pone.0310714.ref009","doi-asserted-by":"crossref","first-page":"e8647","DOI":"10.1371\/journal.pone.0008647","article-title":"A Duchenne Muscular Dystrophy Gene Hot Spot Mutation in Dystrophin-Deficient Cavalier King Charles Spaniels Is Amenable to Exon 51 Skipping","volume":"5","author":"GL Walmsley","year":"2010","journal-title":"PLoS ONE"},{"issue":"1","key":"pone.0310714.ref010","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1089\/humc.2015.006","article-title":"Duchenne Muscular Dystrophy Gene Therapy in the Canine Model","volume":"26","author":"D. 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