Ribonuclease Protection
Ribonuclease protection is a procedure that uses uniformly labeled RNA probes to analyze sample RNA. In this case, probes are chosen to fall entirely within the coding region, so they are only digested if no homologous RNA is present. Because the probes are uniformly labeled, the sensitivity of this technique is much higher than that for S-1 mapping. An excess of probe is mixed with the sample to be analyzed, and the hybrids are digested with RNase A, which will digest only ssRNA. The amount of probe protected from digestion (because it has hybridized with target RNA) is quantified by denaturing gel electrophoresis followed by autoradiography or by running on an automated sequencer. One of the strengths of this technique is that multiple probes can be added to a single sample, provided that they are of different sizes. A single reaction can thus give quantitative information on many different RNA species.
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In RNase Protection, an excess of labeled probe is hybridized to the mRNA pool. Digestion with RNase followed by gel electrophoresis (Probe + mRNA) provides quantitation of the amount of probe complementary mRNA expressed. After digestion in the absence of mRNA (Probe - mRNA) no probe remains. |
Ribonuclease Protection
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