Protocol for a minigene splice assay using the pET01 vector
Aberrant splicing plays a major role in hereditary disorders, yet characterizing molecular effects of splice variants poses challenges. Here, we present a protocol for an in vitro minigene splice assay using the pET01 vector. We describe steps for assay design, minigene plasmid cloning, transfection, RNA isolation, and cDNA synthesis. We also detail procedures for quantitative capillary electrophoresis and optional subcloning. This protocol is useful when patient RNA is unavailable or target genes or isoforms are not expressed in accessible tissues.
Authors
- M. Joan Curcio (ORCID: https://orcid.org/0000-0001-5361-3909)
- Barbara Vona (ORCID: https://orcid.org/0000-0002-6719-3447)
- Fritz Benseler (ORCID: https://orcid.org/0009-0006-3846-9447)
- Nils Brose (ORCID: https://orcid.org/0000-0003-0938-8534)
- Daniel Owrang
- Hannah Andreae
- Sahar Esmaeelpour
- Friederike Jahnke
Institutions
- European Neuroscience Institute Göttingen (DE)
- Max Planck Institute for Multidisciplinary Sciences (DE)
- University of Göttingen (DE)
Publication Details
- Journal
- STAR Protocols
- Published
- 2025-06-18
- DOI
- https://doi.org/10.1016/j.xpro.2025.103908
- Citations
- 4
- Primary Topic
- RNA Research and Splicing
- Type
- article
- Field-Weighted Citation Impact
- 3.22
Funders
- Università degli Studi di Napoli Federico II
- Universitätsmedizin Göttingen
- Deutsche Forschungsgemeinschaft
- Else Kröner-Fresenius-Stiftung
- Erasmus+