RNAscope in situ hybridization-based method for detecting RNA expression in vitro.

Facioscapulohumeral muscular dystrophy (FSHD) is among the most common forms of muscular dystrophy. FSHD is caused by aberrant expression of the toxic gene in muscle. Detecting endogenous in patient tissue using conventional methods can be challenging, due to the low level of expression. Therefore, developing simple and trustworthy detection methods is an important need in the FSHD field. Here, we describe such a method, which uses the RNAscope assay, an RNA in situ hybridization (ISH) technology. We show that a custom-designed RNAscope assay can detect overexpressed mRNA in transfected HEK293 cells and endogenous mRNA in FSHD patient-derived myotubes. The RNAscope assay was highly sensitive for tracking reductions in mRNA following treatment with our therapeutic mi405 microRNA, suggesting that RNAscope-based expression assays could be developed as a prospective outcome measure in therapy trials. This study could set the stage for optimizing and developing a new, rapid RNA ISH-based molecular diagnostic assay for future clinical use in the FSHD field.