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Nuclease Protection Assays: The Basics


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What is a Nuclease Protection Assay?

Nuclease protection assays (NPAs), including both ribonuclease protection assays (RPAs) and S1 nuclease assays, are an extremely sensitive method for the detection, quantitation and mapping of specific RNAs in a complex mixture of total cellular RNA. The basis of NPAs is a solution hybridization of a single-stranded, discrete sized antisense probe(s) to an RNA sample (see Figure 1). The small volume solution hybridization is far more efficient than more common membrane-based hybridization, and can accommodate up to 100 g of total or poly(A) RNA. After hybridization, any remaining unhybridized probe and sample RNA are removed by digestion with a mixture of nucleases. Then, in a single step reaction, the nucleases are inactivated and the remaining probe:target hybrids are precipitated. These products are separated on a denaturing polyacrylamide gel and are visualized by autoradiography. If nonisotopic probes are used, samples are visualized by transferring the gel to a membrane and performing secondary detection.

Figure 1. Detection of Specific mRNA Species Using a Nuclease Protection Assay.


NPAs are the method of choice for the simultaneous detection of several RNA s
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