S1 nuclease analysis of alternatively spliced mRNA

Martin Lützelberger, Jørgen Kjems

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    Abstract

    The characterization of alternatively spliced RNA is a frequently performed task in the molecular biology laboratory. Several methods have been established to characterize specific transcripts, of which microarrays, northern analysis, RT-PCR and nuclease protection assays are the most frequently performed methods in the laboratory. Here, we describe the analysis of alternatively spliced RNA by using 5(')-end labelled DNA oligonucleotide probes and S1 nuclease. The method is sensitive, allowing detection of as little as a few hundred femtograms of a specific RNA, and useful for the quantitation of alternatively spliced mRNA isoforms. Because of its insensitivity towards RNA secondary structures and partially degraded RNA, it may perform better in the quantitation of RNA than northern analysis or RT-PCR, especially when long transcripts are studied.
    Original languageEnglish
    JournalMethods in Molecular Biology
    Volume703
    Pages (from-to)161-71
    Number of pages11
    ISSN1064-3745
    DOIs
    Publication statusPublished - 1 Jan 2011

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