RNase protection assay

Ying Jun Ma, Gregory Dissen, Florence Rage, Sergio Ojeda

    Research output: Contribution to journalArticle

    36 Citations (Scopus)

    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

    Fingerprint

    Ribonucleases
    Assays
    Messenger RNA
    Complementary RNA
    RNA
    Tissue
    RNA Probes
    Antisense RNA
    Endopeptidase K
    Phenol
    Digestion

    ASJC Scopus subject areas

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

    Cite this

    RNase protection assay. / Ma, Ying Jun; Dissen, Gregory; Rage, Florence; Ojeda, Sergio.

    In: Methods: A Companion to Methods in Enzymology, Vol. 10, No. 3, 12.1996, p. 273-278.

    Research output: Contribution to journalArticle

    Ma, Ying Jun ; Dissen, Gregory ; Rage, Florence ; Ojeda, Sergio. / RNase protection assay. In: Methods: A Companion to Methods in Enzymology. 1996 ; Vol. 10, No. 3. pp. 273-278.
    @article{46e4169b40c1488b9a2186b7fbf1b51b,
    title = "RNase protection assay",
    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.",
    author = "Ma, {Ying Jun} and Gregory Dissen and Florence Rage and Sergio Ojeda",
    year = "1996",
    month = "12",
    doi = "10.1006/meth.1996.0102",
    language = "English (US)",
    volume = "10",
    pages = "273--278",
    journal = "ImmunoMethods",
    issn = "1046-2023",
    publisher = "Academic Press Inc.",
    number = "3",

    }

    TY - JOUR

    T1 - RNase protection assay

    AU - Ma, Ying Jun

    AU - Dissen, Gregory

    AU - Rage, Florence

    AU - Ojeda, Sergio

    PY - 1996/12

    Y1 - 1996/12

    N2 - 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.

    AB - 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.

    UR - http://www.scopus.com/inward/record.url?scp=0030560922&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=0030560922&partnerID=8YFLogxK

    U2 - 10.1006/meth.1996.0102

    DO - 10.1006/meth.1996.0102

    M3 - Article

    VL - 10

    SP - 273

    EP - 278

    JO - ImmunoMethods

    JF - ImmunoMethods

    SN - 1046-2023

    IS - 3

    ER -