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

저자: Hassan T, Smith SG, Gaughan K, Oglesby IK, O'Neill S, McElvaney NG, Greene CM.

저널: Nucleic Acids Res. 2013 Apr 1;41(6):e71.

사용한 제품: Dynabeads M-280 Streptavidin (Cat# 11205D, 11206D, 60210)

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.

제품 사용법

Streptavidin bead preparation and miRNA:AAT mRNA pulldown

Streptavidin bead preparation and miRNA:AAT mRNA pulldown Immobilization of 2000 pmol/ml of biotinylated capture oligonucleotide with 10 mg of streptavidin beads (Dynabeads M-280 Streptavidin) was performed according to the manufacturer’s instructions. Oligo-prepared beads (10 mg) were incubated with 1 ml of binding buffer [20 mM Tris–HCl (pH 7.5), 2mM EDTA, 1M LiCl] and mixed with an equal volume of formaldehyde-treated cell lysates for 30 min at room temperature for adequate annealing. The beads were washed twice using washing buffer [10 mM Tris–HCl (pH 7.5), 1mM EDTA, 0.15mM LiCl] and AAT mRNA:miRNA complexes were captured using a Dynal magnet. The beads were suspended in 10mM Tris–HCl (pH 7.5) before heat treatment at 80 C for 5 min to reverse the interaction between the biotin-labelled DNA:mRNA:miRNA complexes and the magnetic beads. Cross-linked nucleic acids and proteins were treated with proteinase K (Sigma-Aldrich) for digestion of RBPs and remaining DNase for 1 h at 42 C. Next incubation at 65 C for 20 min was performed to fully reverse the cross-linkages. Validation was performed to confirm the presence of AAT mRNA and true target miRNAs and to rule out the presence of other mRNAs and miRNAs by qRT-PCR and Taqman miRNA assay. To determine the enrichment of the mRNA of interest compared with the expression of highly abundant mRNAs that may non-specifically bind to the oligo-beads, the 2   Ct method was used to normalize the Cq values against baseline expression of mRNAs in cell lysates.