Identification of qPCR reference genes suitable for normalizing gene expression in the mdx mouse model of Duchenne muscular dystrophy

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.mdx小鼠（mdx mouse）是目前应用最为广泛的杜氏肌营养不良症（Duchenne muscular dystrophy）人类疾病动物模型，对该动物肌肉组织的基因表达开展定量PCR（quantitative PCR）分析，在该病发病机制、疾病进程研究以及潜在治疗干预手段的评估中均发挥关键作用。对合适的稳定表达参考基因进行归一化校正，是实现精准定量检测的必要前提，但筛选此类基因颇具挑战：健康与营养不良性肌肉的转录环境差异显著，且随疾病进程和肌肉使用状态进一步改变，这意味着不存在任何单一基因或基因组合可在所有实验比较场景中保持稳定表达。尽管该动物模型已是经典研究模型，此问题至今仍未得到解决。因此，本研究旨在全面评估健康与营养不良性小鼠肌肉中参考基因的适用性，筛选出适用于特定实验比较场景的参考基因，并探究是否存在一套可通用的参考基因集。本研究收集了大规模样本，包含来自健康小鼠和mdx小鼠的多种肌肉组织（包括胫骨前肌、膈肌与心肌），覆盖3个与疾病进程相关的年龄组，同时选取了16个候选参考基因（FBXO38、FBXW2、MON2、ZFP91、HTATSF1、GAPDH、ACTB、18S、CDC40、SDHA、RPL13a、CSNK2A2、AP3D1、PAK1IP1、B2M及HPRT1），并通过geNorm、BestKeeper及Normfinder算法筛选出可在多种比较场景中保持稳定表达的基因。研究结果显示，不存在可在所有条件下稳定表达的单一基因，但由RPL13a、CSNK2A2、AP3D1及常用基因ACTB组成的归一化因子，似乎可适用于健康与营养不良性小鼠肌肉的基因表达归一化，且不受肌肉类型或动物年龄的影响。本研究进一步发现，其他热门参考基因（包括GAPDH）显著与疾病状态或肌肉类型相关。本研究证实了基于实验筛选参考基因的重要性，可为希望开展mdx小鼠基因表达研究的研究者提供宝贵的参考资源。