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Alternative polyadenylation (APA) represents a pivotal post-transcriptional mechanism, exerting a significant influence on gene expression through the generation of diverse 3′-end RNA isoforms. The present protocol delineates a streamlined approach for modulating APA using antisense oligonucleotides (ASOs), with a focus on their design, application, and analytical evaluation. By targeting specific polyadenylation signals, ASOs enable the reprogramming of isoform proportions, as demonstrated by the regulation of immunoglobulin heavy chain transcript isoforms. The protocol delineates the sequence of steps involved in the method, commencing with the design and validation of ASOs, followed by treatment of cells, RNA purification, and quantitative PCR-based isoform analysis. Key factors, such as ASO design and treatment optimization, are considered to ensure reproducibility and robustness. This workflow offers a robust platform to assess the therapeutic potential of ASOs in APA modulation across various cell types.

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Modifying 3′-End RNA Isoforms Using Antisense Oligonucleotides

  • 实验方案

  • 发表日期：

    2025年

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  • Antoine Praité,
  • Catherine Horiot,
  • Anne Marchalot,
  • Sandy Al Hayek,
  • Laurent Delpy

摘要

Alternative polyadenylation (APA) represents a pivotal post-transcriptional mechanism, exerting a significant influence on gene expression through the generation of diverse 3′-end RNA isoforms. The present protocol delineates a streamlined approach for modulating APA using antisense oligonucleotides (ASOs), with a focus on their design, application, and analytical evaluation. By targeting specific polyadenylation signals, ASOs enable the reprogramming of isoform proportions, as demonstrated by the regulation of immunoglobulin heavy chain transcript isoforms. The protocol delineates the sequence of steps involved in the method, commencing with the design and validation of ASOs, followed by treatment of cells, RNA purification, and quantitative PCR-based isoform analysis. Key factors, such as ASO design and treatment optimization, are considered to ensure reproducibility and robustness. This workflow offers a robust platform to assess the therapeutic potential of ASOs in APA modulation across various cell types.