Accurate Dystrophin Quantification in Mouse Tissue; Identification of New and Evaluation of Existing Methods.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder primarily affecting males. This disorder is caused by mutations in the DMD gene that abolish dystrophin protein function. Many therapeutic approaches for DMD aim at recovery of the dystrophin protein in muscle fibers of affected patients, rendering accurate dystrophin quantification important. Several methods have been reported to detect and quantify dystrophin restoration in preclinical and clinical trials. We here evaluated the applicability of dystrophin specific enzyme-linked immunosorbent assays (ELISA) and a TaqMan protein assay, benchmarking them against Western blotting analysis. Despite numerous optimization attempts, in our hands the background signals in the ELISA and TaqMan protein assays were too high to allow dystrophin quantification. By contrast, the Western blot approach was able to detect dystrophin levels as low as 0.2% in a reproducible manner. We provide a Western blot protocol that allows sensitive and accurate dystrophin quantification in preclinical studies.