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The RNase protection assay.

Michael F Carey, Craig L Peterson, Stephen T Smale

    Cold Spring Harbor Protocols
    |March 5, 2013
    PubMed
    Summary
    This summary is machine-generated.

    The RNase protection assay precisely locates transcription start sites using a radiolabeled RNA probe. This method identifies the exact start site by measuring the protected RNA fragment after nuclease digestion.

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    Area of Science:

    • Molecular Biology
    • Genetics

    Background:

    • Transcription start-site localization is crucial for understanding gene regulation.
    • Existing methods may lack sensitivity or precision.

    Purpose of the Study:

    • To describe and validate the RNase protection assay for accurate transcription start-site mapping.

    Main Methods:

    • Synthesize a radiolabeled RNA probe using bacteriophage RNA polymerase and a plasmid vector containing the target genomic DNA.
    • Anneal the probe to cellular mRNA and digest with RNases (RNase A and/or T1).
    • Analyze the size of the RNase-resistant RNA fragment via denaturing polyacrylamide gel electrophoresis.

    Main Results:

    • The assay provides sensitive and precise localization of transcription start sites.

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  • The length of the protected RNA fragment directly correlates with the distance from the probe's 5' end to the transcription start site.

    • Conclusions:

    • The RNase protection assay is a reliable method for determining transcription start sites.
    • This technique aids in the detailed analysis of gene expression and regulation.