A Sensitive Alternative for MicroRNA In Situ Hybridizations Using Probes of 2'-O-Methyl RNA + LNA

Martin Jensen Søe, Trine Møller, Martin Dufva, Kim Holmstrøm

    Research output: Contribution to journalJournal articleResearchpeer-review

    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.
    Original languageEnglish
    JournalJournal of Histochemistry and Cytochemistry
    Volume59
    Issue number7
    Pages (from-to)661-672
    ISSN0022-1554
    DOIs
    Publication statusPublished - 2011

    Keywords

    • MicroRNA-205
    • Yeast RNA
    • 2′-O-methyl RNA
    • MicroRNA-138
    • In situ hybridization
    • MicroRNA, locked nucleic acids
    • Urea
    • Formalin-fixed paraffin-embedded

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