title: A sensitive alternative for microRNA in situ hybridizations using probes of 2'-O-methyl RNA + LNA.
abstract: The use of short, high-affinity probes consisting of a combination of DNA and locked nucleic acid (LNA) has enabled the specific detection of microRNAs (miRNAs) by in situ hybridization (ISH). However, detection of low-copy number miRNAs is still not always possible. Here the authors show that probes consisting of 2'-O-methyl RNAs (2OMe) and LNA at every third base (2:1 ratio), under optimized hybridization conditions, excluding yeast RNA from the hybridization buffer, can provide superior performance in detection of miRNA targets in terms of sensitivity and signal-to-noise ratio compared to DNA + LNA probes. Furthermore, they show that hybridizations can be performed in buffers of 4M urea instead of 50% formamide, thereby yielding an equally specific but nontoxic assay. The use of 2OMe + LNA-based probes and the optimized ISH assay enable simple and fast detection of low-copy number miRNA targets, such as miR-130a in mouse brain.
authors.affiliation: Bioneer A/S, Hørsholm, Denmark (MJS,TM,KH).
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Søe
authors.forename: Trine
authors.initials: T
authors.lastname: Møller
authors.forename: Martin
authors.initials: M
authors.lastname: Dufva
authors.forename: Kim
authors.initials: K
authors.lastname: Holmstrøm
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D001923:Brain Chemistry; D017403:In Situ Hybridization / Q000379:methods*; D008321:Mammary Glands, Animal / Q000737:chemistry; D008745:Methylation; D051379:Mice; D035683:MicroRNAs / Q000032:analysis*; D009206:Myocardium / Q000737:chemistry; D015341:Nucleic Acid Probes / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D012680:Sensitivity and Specificity
chemical_list: D035683:MicroRNAs; D015341:Nucleic Acid Probes; D009841:Oligonucleotides; C477371:locked nucleic acid; D012313:RNA