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Updated: Oct 24, 2025

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA
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Antisense Oligo Pulldown of Circular RNA for Downstream Analysis.

Debojyoti Das1,2, Aniruddha Das1,2, Amaresh C Panda1

  • 1Institute of Life Sciences, Nalco Square, Bhubaneswar, Odisha, India.

Bio-Protocol
|August 16, 2021
PubMed
Summary

This study presents a quick and easy method to capture specific circular RNAs (circRNAs) using biotin-labeled antisense oligos. This technique helps identify and validate interactions between circRNAs, microRNAs (miRNAs), and RNA-binding proteins (RBPs).

Keywords:
Antisense oligoBiotin pulldownCircRNA pulldownCircRNAsRNA-binding proteinsRT-qPCRmiRNA

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Circular RNAs (circRNAs) are noncoding RNA molecules regulating gene expression by binding microRNAs (miRNAs) and RNA-binding proteins (RBPs).
  • Computational tools predict circRNA interactions, but experimental validation under physiological conditions is limited.
  • Existing biochemical assays for capturing circRNA-miRNA-RBP complexes have limitations.

Purpose of the Study:

  • To detail a protocol for capturing specific circRNAs using a biotin-labeled antisense oligonucleotide (ASO) pulldown method.
  • To validate the interaction of circRNAs with specific miRNAs and RBPs.
  • To provide a reliable method for studying circRNA regulatory functions.

Main Methods:

  • A biotin-labeled antisense oligonucleotide (ASO) targeting the circRNA backsplice junction was used for capture.
  • Streptavidin-conjugated magnetic beads facilitated the pulldown of the targeted circRNA.
  • Reverse transcription quantitative PCR (RT-qPCR) analyzed circRNA enrichment.
  • miRNA RT-qPCR and western blotting confirmed miRNA and RBP associations.

Main Results:

  • The developed ASO pulldown method successfully captured the target circRNA (circHipk2).
  • RT-qPCR confirmed specific enrichment of the target circRNA.
  • Coupled assays validated the interaction of predicted miRNAs and RBPs with the captured circRNA.

Conclusions:

  • The biotin-labeled ASO pulldown assay is a quick, easy, and effective method for capturing specific circRNAs.
  • This technique is valuable for identifying and validating interactions between circRNAs, miRNAs, and RBPs.
  • The protocol facilitates the study of circRNA functions in cellular physiology.