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Et reporterbaseret cellulært assay til overvågning af splejsningseffektivitet
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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
Denne protokol beskriver et minigen reporter-assay for at overvåge virkningen af 5'-splejsningsstedsmutationer på splejsning og udvikler suppressor U1 snRNA til redning af mutationsinduceret splejsningshæmning. Reporter- og suppressoren U1 snRNA-konstruktioner udtrykkes i HeLa-celler, og splejsning analyseres ved primerudvidelse eller RT-PCR.
