Abstract
The mdx mouse is the most widely-used animal model of the human disease Duchenne muscular dystrophy, and quantitative PCR analysis of gene expression in the muscles of this animal plays a key role in the study of pathogenesis and disease progression and in evaluation of potential therapeutic interventions. Normalization to appropriate stably-expressed reference genes is essential for accurate quantitative measurement, but determination of such genes is challenging: healthy and dystrophic muscles present very different transcriptional environments, further altering with disease progression and muscle use, raising the possibility that no single gene or combination of genes may be stable under all experimental comparative scenarios. Despite the pedigree of this animal model, this problem remains unaddressed. The aim of this work was therefore to comprehensively assess reference gene suitability in the muscles of healthy and dystrophic mice, identifying reference genes appropriate for specific experimental comparisons, and determining whether an essentially universally-applicable set of genes exists. Using a large sample collection comprising multiple muscles (including the tibialis anterior, diaphragm and heart muscles) taken from healthy and mdx mice at three disease-relevant ages, and a panel of sixteen candidate reference genes (FBXO38, FBXW2, MON2, ZFP91, HTATSF1, GAPDH, ACTB, 18S, CDC40, SDHA, RPL13a, CSNK2A2, AP3D1, PAK1IP1, B2M and HPRT1), we used the geNorm, BestKeeper and Normfinder algorithms to identify genes that were stable under multiple possible comparative scenarios. We reveal that no single gene is stable under all conditions, but a normalization factor derived from multiple genes (RPL13a, CSNK2A2, AP3D1 and the widely-used ACTB) appears suitable for normalizing gene expression in both healthy and dystrophic mouse muscle regardless of muscle type or animal age. We further show that other popular reference genes, including GAPDH, are markedly disease- or muscle-type correlated. This study demonstrates the importance of empirical reference gene identification, and should serve as a valuable resource for investigators wishing to study gene expression in mdx mice.
Highlights
The X-linked muscle-wasting disease Duchenne muscular dystrophy (DMD) affects roughly 1 in 5000 new born boys [1], and is the most common fatal genetic condition diagnosed in childhood
Dystrophic muscle samples were obtained from male mdx mice bred under UK Home Office Project Licence PPL 70/7777 together with healthy strainmatched male C57/Bl10 wild type (WT) samples from mice bred under the same licence
Distribution of within-gene Cq values for our remaining thirteen candidates were lowest in AP3D1 and CSNK2A2, and highest in FBXW2 (S1 Table)
Summary
The X-linked muscle-wasting disease Duchenne muscular dystrophy (DMD) affects roughly 1 in 5000 new born boys [1], and is the most common fatal genetic condition diagnosed in childhood. While the condition is presently incurable, DMD remains a field of active research: several different approaches aimed at dystrophin restoration are currently under investigation or entering therapeutic trials [3, 4]. Such research is aided by animal models, and multiple models of this disease exist, including mouse [5], rat [6, 7], rabbit [8], dog [9,10,11] and pig [12, 13]. Regardless of species, assessment of the consequences of insufficient dystrophin -and more critically, the extent of therapeutic dystrophin restoration- utilises multiple investigative avenues, from whole animal and whole muscle physiological studies, to gross histology and immunostaining, to quantitative measures of gene expression at the protein and mRNA level
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