Putker Kayleigh, Schneider Anne-Fleur, Van De Vijver Davy, Hildyard John, Aartsma-Rus Annemieke, van Putten Maaike
Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Department of Clinical Sciences and Services, Comparative Neuromuscular Diseases Laboratory, Royal Veterinary College, London, United Kingdom.
PLoS One. 2025 Feb 25;20(2):e0318944. doi: 10.1371/journal.pone.0318944. eCollection 2025.
Duchenne muscular dystrophy (DMD) is an X-linked disorder that is caused by mutations in the DMD gene, leading to progressive muscle wasting and weakness. There is currently no cure for DMD. The BL10-mdx mouse is the most commonly used model in preclinical DMD studies, but it exhibits a mild disease phenotype compared to DMD patients, limiting research translatability. The newer D2-mdx mouse has a more severe phenotype at an early age and may better recapitulate human disease. To compare these mouse models on a transcriptional level with quantitative RT-PCR, stable and reliable reference genes are indispensable. We aimed to evaluate the stability and reliability of a panel of nine candidate reference genes (Actb, Ap3d1, Gapdh, Hmbs, Htatsf1, Pak1ip1, Rpl13a, Sdha and Zfp91) in the gastrocnemius, diaphragm and heart of mice from both strains and their corresponding wild types aged 4 to 52 weeks. Data was analyzed using geNorm, BestKeeper, deltaCt and NormFinder. We found that Htatsf1, Pak1ip1 and Zfp91 are suitable reference genes for normalization of gene expression in dystrophic and healthy mice, regardless of the tissue type or age. In our hands, Actb, Gapdh and Rpl13a were not suitable as reference genes, exhibiting tissue-, age-, or disease specific changes in expression. This study highlights the importance of the selection of suitable reference genes, as their stability can differ between specific experimental setups.
杜氏肌营养不良症（DMD）是一种X连锁疾病，由DMD基因突变引起，导致进行性肌肉萎缩和无力。目前尚无治愈DMD的方法。BL10-mdx小鼠是临床前DMD研究中最常用的模型，但与DMD患者相比，它表现出轻度疾病表型，限制了研究的可转化性。较新的D2-mdx小鼠在幼年时具有更严重的表型，可能更能模拟人类疾病。为了通过定量逆转录聚合酶链反应在转录水平上比较这些小鼠模型，稳定可靠的内参基因是必不可少的。我们旨在评估一组9个候选内参基因（Actb、Ap3d1、Gapdh、Hmbs、Htatsf1、Pak1ip1、Rpl13a、Sdha和Zfp91）在4至52周龄的两种品系小鼠及其相应野生型的腓肠肌、膈肌和心脏中的稳定性和可靠性。使用geNorm、BestKeeper、deltaCt和NormFinder分析数据。我们发现，无论组织类型或年龄如何，Htatsf1、Pak1ip1和Zfp91都是用于营养不良和健康小鼠基因表达标准化的合适内参基因。在我们的研究中，Actb、Gapdh和Rpl13a不适合作为内参基因，它们在表达上表现出组织、年龄或疾病特异性变化。这项研究强调了选择合适内参基因的重要性，因为它们的稳定性在特定实验设置之间可能会有所不同。
