Finding the best genes for normalizing qPCR data in a dog model of DMD
What is it about?
The muscle wasting disease Duchenne muscular dystrophy (DMD) is invariably fatal, has no cure, and affects 1 in 5000 newborn boys. Study of this disease (and response to treatments) is important, and one method that is widely used is the measurement of gene expression (how active a given gene is) via quantitative polymerase chain reaction (qPCR) methods. This process involves a lot of steps, each of which could potentially be a source of error, meaning it is very important to have some form of internal standard for each sample. In the case of qPCR, this is a gene (or genes) that you know does NOT change behavior between your samples, so that any changes you do observe can be safely explained as preparation error, and accounted for. Finding such 'reference genes' is not easy, and there is no single gene that works for every situation. Finding a stable reference gene or genes is particularly important for study of diseases like DMD where the disease brings about broad changes in gene behavior, and here we have used a dog model of this disease to discover a set of three reference genes that are universally suitable in both healthy and diseased muscles, regardless of age or muscle studied. This will be of great benefit to studies using this dog model, as it allows all muscles to be compared, at all stages of disease (or all stages of treatment).
Why is it important?
Our findings not only identify good reference genes for this animal model of a fatal human disease: they also show that these reference genes are essentially universally suitable within this model. This greatly increases the utility of gene expression studies using this model of DMD, and more importantly shows that such broadly-suitable panels of genes can exist, and that similar panels might be found in other animal models (or indeed human patients). In a more general sense, our work also explains in relatively accessible language exactly why taking the effort to determine good reference genes is important, and how such determinations should be performed. qPCR is easy to do, but considerably harder to do well: we hope our work encourages others to adopt a more rigorous approach to reference gene selection.