title: Isolation and identification of cell-specific microRNAs targeting a messenger RNA using a biotinylated anti-sense oligonucleotide capture affinity technique.
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.
authors: Respiratory Research Division, Department of Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Republic of Ireland.
authors: Tidi
authors: T
authors: Hassan
authors: Stephen G J
authors: SG
authors: Smith
authors: Kevin
authors: K
authors: Gaughan
authors: Irene K
authors: IK
authors: Oglesby
authors: Shane
authors: S
authors: O'Neill
authors: Noel G
authors: NG
authors: McElvaney
authors: Catherine M
authors: CM
authors: Greene
mesh_terms: D001665:Binding Sites; D019921:Biotinylation; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics; D035683:MicroRNAs / Q000302:isolation & purification* / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense; D012322:RNA Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053481:Secretory Leukocyte Peptidase Inhibitor / Q000235:genetics; D059467:Transcriptome; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000378:metabolism
chemical_list: D016209:Interleukin-8; D035683:MicroRNAs; D020319:Oligodeoxyribonucleotides, Antisense; D012322:RNA Precursors; D012333:RNA, Messenger; D053481:Secretory Leukocyte Peptidase Inhibitor; D000515:alpha 1-Antitrypsin