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Chapters
- 00:04Introduction
- 00:31Cotransfection of HeLa Cells with the Reporter and U1 Small Nuclear RNA (snRNA) Plasmids
- 02:0032P-Labeling of Oligonucleotides
- 02:52Analysis of the Spliced Reporter Transcripts by Primer Extension
- 03:53Analysis of the Spliced Reporter Transcripts by Fluorescent RT-PCR
- 05:08Analysis of Variant U1 snRNA Expression by RT-qPCR
- 05:38Setup and Running of Urea-PAGE Gels
- 06:27Results: Reporter Based Splicing Efficiency of U1 snRNA Constructs
- 08:09Conclusion
This protocol describes a minigene reporter assay to monitor the impact of 5´-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.
