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Biochemistry

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JoVE Journal Biochemistry
A Reporter Based Cellular Assay for Monitoring Splicing Efficiency
 

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

Article DOI: 10.3791/63014-v • 08:53 min • September 15th, 2021
September 15th, 2021 •
1, 1, 1
1Department of Basic Medical Sciences, College of Medicine-Phoenix, University of Arizona

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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.

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Keywords: Reporter Assay Splicing Efficiency Disease-associated Mutations Exon Splice Sites Splice Site Recognition HeLa Cells Transient Transfection Cell Suspension Molecular Weight Cutoff Size Exclusion Beads RNA Extraction P32 Labeling
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