Department of Clinical Science and Services, Comparative Neuromuscular Diseases Laboratory, Royal Veterinary College, London, UK.
Department of Comparative Biomedical Sciences, Royal Veterinary College, London, UK.
J Neuromuscul Dis. 2018;5(2):177-191. doi: 10.3233/JND-170267.
Dogs with dystrophin-deficient muscular dystrophy are valuable models of the equivalent human disease, Duchenne Muscular Dystrophy (DMD): unlike the mdx mouse, these animals present a disease severity and progression that closely matches that found in human patients. Canine models are however less thoroughly characterised than the established mdx mouse in many aspects, including gene expression. Analysis of expression in muscle plays a key role in the study of DMD, allowing monitoring and assessment of disease progression, evaluation of novel biomarkers and gauging of therapeutic intervention efficacy. Appropriate normalization of expression data via carefully selected reference genes is consequently essential for accurate quantitative assessment. Unlike the expression profile of healthy skeletal muscle, the dystrophic muscle environment is highly dynamic: transcriptional profiles of dystrophic muscle might alter with age, disease progression, disease severity, genetic background and between muscle groups.
The aim of this work was to identify reference genes suitable for normalizing gene expression in healthy and dystrophic dogs under various comparative scenarios.
Using the delta-E50 MD canine model of DMD, we assessed a panel of candidate reference genes for stability of expression across healthy and dystrophic animals, at different ages and in different muscle groups.
We show that the genes HPRT1, SDHA and RPL13a appear universally suitable for normalizing gene expression in healthy and dystrophic canine muscle, while other putative reference genes are exceptionally poor, and in the case of B2M, actively disease-correlated.
Our findings suggest consistent cross-sample normalization is possible even throughout the dynamic progression of dystrophic pathology, and furthermore highlight the importance of empirical determination of suitable reference genes for neuromuscular diseases.
患有肌营养不良症的狗是人类疾病杜氏肌营养不良症（DMD）的宝贵模型：与 mdx 小鼠不同，这些动物的疾病严重程度和进展与人类患者非常相似。然而，与已建立的 mdx 小鼠相比，犬模型在许多方面的特征不如后者，包括基因表达。肌肉表达分析在 DMD 研究中起着关键作用，允许监测和评估疾病进展，评估新的生物标志物，并评估治疗干预的效果。因此，通过精心选择的参考基因对表达数据进行适当的归一化对于准确的定量评估至关重要。与健康骨骼肌的表达谱不同，营养不良的肌肉环境非常动态：营养不良肌肉的转录谱可能随年龄、疾病进展、疾病严重程度、遗传背景和肌肉群之间而改变。
本工作的目的是鉴定适合在各种比较情况下对健康和营养不良犬的基因表达进行归一化的参考基因。
使用 DMD 的 delta-E50 MD 犬模型，我们评估了候选参考基因在不同年龄和不同肌肉群的健康和营养不良动物中的表达稳定性。
我们表明，HPRT1、SDHA 和 RPL13a 这三个基因在健康和营养不良的犬肌肉中普遍适合用于归一化基因表达，而其他假定的参考基因则非常差，在 B2M 的情况下，与疾病呈正相关。
我们的发现表明，即使在营养不良病理的动态进展过程中，也可以进行一致的跨样本归一化，并且进一步强调了为神经肌肉疾病确定合适的参考基因的重要性。
