Background: Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by mutations in the dystrophin gene. DE50-MD dogs are an animal model of DMD used as a final translational model for evaluation of promising treatments. MicroRNA (miR) expressions in the muscle of DE50-MD dogs represent potential biomarkers, but stable reference miRs must first be identified. The aim of this paper was to establish a panel of reference miRs for WT and DE50-MD dogs over a range of ages and muscle groups.
Methods: RNA was extracted from WT and DE50-MD dog (N=6 per genotype) vastus lateralis muscle samples collected longitudinally at 3, 6, 9, 12, 15 and 18 months of age, and from muscles collected post-mortem (N=3 per genotype; cranial tibial, semimembranosus, lateral triceps and diaphragm). 87 RNAs were quantified in a subset of 6-month-old WT and DE50-MD muscles (N=4 per genotype) using the QIAcuity miFinder panel. GeNorm, BestKeeper and Normfinder were used to identify a candidate panel of the 8 most stable small RNAs, which were then quantified in all RNA samples, alongside the commonly used reference RNA snRNA U6.
Results: The most stable miRs of this subset were used to normalise quantities of dystromiRs miR-1, miR-133a and miR-206, and fibromiR miR-214. MicroRNAs miR-191, let-7b, miR-125a and miR-15a were the most stable miRs tested, while snRNA U6 performed poorly. DystromiR expression, normalised to the geometric mean of the panel of reference miRs, was lower for miR-1 and miR-133a in DE50-MD compared to WT muscles, while miR-206 levels did not significantly differ between genotypes. FibromiR miR-214 was 2- to 4-fold higher in DE50-MD versus WT muscles.
Conclusions: A normalisation factor derived from miR-191, let-7b, miR-125a and miR-15a is suitable for normalising miR expression data from WT and DE50-MD muscle over a range of ages and muscle types.
Keywords: DE50-MD; DMD; MicroRNA; RT-qPCR; dog model; reference genes.
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by mutations in the dystrophin gene. DE50-MD dogs are a canine model of DMD that manifest the disease in a very similar way to human DMD patients. To further our understanding of DMD and to identify potential markers of an improvement in the condition following treatment, we aimed to quantify the expression of several microRNAs (miRs) in muscle samples. miRs are small RNA molecules that regulate the expression of genes, whose expression is often altered in DMD patients. To ensure reliable quantification, we must first identify miRs that are stably expressed in muscle to use as a normalisation factor that will account for any variation that may be introduced during the experimental process. We enrolled 6 healthy and 6 DE50-MD dogs and collected muscle samples at 3-monthly intervals from 3- to 18-months of age. RNA was extracted from these samples and a large panel of potential reference RNAs were quantified. We used 3 different algorithms to test the stability of each of these RNAs in our sample pool: GeNorm, BestKeeper and Normfinder. MicroRNAs miR-191, let-7b, miR-125a and miR-15a were the most stable miRs tested. These 4 miRs were used to normalise the amount of several miRs of interest to DMD. Expression of miRs of interest miR-1 and miR-133a, when normalised to the top 4 reference RNAs, were reduced in DE50-MD compared to healthy dogs, while miR-206 levels did not differ between groups, and miR-214 levels were increased. These results are consistent with findings in human DMD patients. We conclude that normalisation to expression of miRs-191, let-7b, miR-125a and miR-15a is suitable for reliable quantification of miR data from DE50-MD and healthy dog muscle over a range of ages.
Copyright: © 2024 Riddell DO et al.