Isolation and identification of cell-specific microRNAs targeting a messenger RNA using a biotinylated anti-sense oligonucleotide capture affinity technique

Hassan Tidi Maharani, Stephen G J Smith, Kevin Gaughan, Irene K. Oglesby, Shane O'Neill, Noel G. McElvaney, Catherine M. Greene

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that regulate expression by translational repression or messenger RNA (mRNA) degradation. Although numerous bioinformatic prediction models exist to identify miRNA-mRNA interactions, experimental validation of bona fide interactions can be difficult and laborious. Few methods can comprehensively identify miRNAs that target a single mRNA. We have developed an experimental approach to search for miRNAs targeting any mRNA using a capture affinity assay involving a biotinylated DNA anti-sense oligonucleotide. This method identifies miRNAs targeting the full length of the mRNA. The method was tested using three separate mRNA targets: alpha-1 antitrypsin (AAT) mRNA, interleukin-8 mRNA and secretory leucoprotease inhibitor mRNA. AAT mRNA-specific and total miRNAs from three different cell lines (monocytic THP-1, bronchial epithelial 16HBE14o- and liver HepG2 cells) were profiled, and validation studies revealed that AAT mRNA-specific miRNAs functionally target the AAT mRNA in a cell-specific manner, providing the first evidence of innate miRNAs selectively targeting and modulating AAT mRNA expression. Interleukin-8 and secretory leucoprotease inhibitor mRNAs and their cognate miRNAs were also successfully captured using this approach. This is a simple and an efficient method to potentially identify miRNAs targeting sequences within the full length of a given mRNA transcript.

Original languageEnglish
JournalNucleic Acids Research
Volume41
Issue number6
DOIs
Publication statusPublished - Apr 2013
Externally publishedYes

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Cell Separation
Antisense Oligonucleotides
MicroRNAs
Messenger RNA
alpha 1-Antitrypsin
Secretory Leukocyte Peptidase Inhibitor
Interleukin-8
Small Untranslated RNA
Validation Studies
RNA Stability
Hep G2 Cells
Computational Biology

ASJC Scopus subject areas

  • Genetics

Cite this

Isolation and identification of cell-specific microRNAs targeting a messenger RNA using a biotinylated anti-sense oligonucleotide capture affinity technique. / Tidi Maharani, Hassan; Smith, Stephen G J; Gaughan, Kevin; Oglesby, Irene K.; O'Neill, Shane; McElvaney, Noel G.; Greene, Catherine M.

In: Nucleic Acids Research, Vol. 41, No. 6, 04.2013.

Research output: Contribution to journalArticle

Tidi Maharani, Hassan ; Smith, Stephen G J ; Gaughan, Kevin ; Oglesby, Irene K. ; O'Neill, Shane ; McElvaney, Noel G. ; Greene, Catherine M. / Isolation and identification of cell-specific microRNAs targeting a messenger RNA using a biotinylated anti-sense oligonucleotide capture affinity technique. In: Nucleic Acids Research. 2013 ; Vol. 41, No. 6.
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