RNase protection assay

Ying Jun Ma, Gregory A. Dissen, Florence Rage, Sergio R. Ojeda

    Research output: Contribution to journalArticlepeer-review

    36 Scopus citations

    Abstract

    The RNase protection assay is a highly sensitive technique developed to detect and measure the abundance of specific mRNAs in samples of total cellular RNA. The assay utilizes in vitro transcribed 32P-labeled antisense RNA probes that are hybridized in solution to their complementary cellular mRNAs. This is followed by digestion of nonhybridizing (single-stranded) RNA species with RNases, removal of the RNases by treatment with proteinase K, phenol extraction of the cRNA:mRNA complexes, and electrophoretic isolation of the hybridizing cRNA fragments. Since one can synthesize 'sense' mRNAs having the same sequence as the target cellular mRNA, appropriate standard curves can be generated and used to quantitate the changes in tissue mRNA levels. Because the assay requires perfect sequence complementarity for full protection, it not only serves as a quantitative tool but also provides conclusive evidence for the existence of a specific mRNA in a given tissue. The procedure described here is a modification of that originally described by M. Gilman.

    Original languageEnglish (US)
    Pages (from-to)273-278
    Number of pages6
    JournalMethods: A Companion to Methods in Enzymology
    Volume10
    Issue number3
    DOIs
    StatePublished - Dec 1996

    ASJC Scopus subject areas

    • Molecular Biology
    • Biochemistry, Genetics and Molecular Biology(all)

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