Protocol for a minigene splice assay using the pET01 vector

Aberrant splicing is recognized as a key contributor of hereditary disorders, yet characterizing the molecular effects of splice variants is an important task that poses several challenges. Here, we present a protocol for an in vitro splice assay using a minigene approach, which is especially useful when patient samples are not available for RNA analysis or when target genes or isoforms are not expressed in accessible tissues for direct analysis. We describe steps for assay design, including the cloning of minigene plasmids and subsequent transfection, followed by RNA isolation and cDNA synthesis. We also provide details on quantitative fragment analysis using capillary electrophoresis and optional subcloning to facilitate optimal sequencing of multiple splicing products.