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Een op reporter gebaseerde cellulaire test voor het bewaken van splicing-efficiëntie
JoVE 신문
생화학
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JoVE 신문 생화학
A Reporter Based Cellular Assay for Monitoring Splicing Efficiency
DOI:

08:53 min

September 15, 2021

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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

Summary

자동 번역

Dit protocol beschrijft een minigene reporter assay om de impact van 5'-splice site mutaties op splicing te monitoren en ontwikkelt suppressor U1 snRNA voor de redding van mutatie-geïnduceerde splicing remming. De reporter en suppressor U1 snRNA constructen worden uitgedrukt in HeLa cellen, en splicing wordt geanalyseerd door primer extensie of RT-PCR.

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