Researchers looking for highly sensitive RNA quantitation and the ability to multiplex often turn to the ribonuclease protection assay (RPA). In an RPA, the RNA sample is incubated with labeled RNA probes complementary to specific target sequences. After the target sequences and probes hybridize, the sample is treated with ribonuclease to degrade any unhybridized probe, and the "protected" probe/target hybrids are separated on a polyacrylamide gel, detected, and quantitated.
Unlike Northern blots, which require blot stripping and reprobing to detect multiple targets, RPAs let the user detect multiple targets simultaneously in the same reaction tube by varying the size of the different probes used, thereby greatly increasing the assay's speed. Unlike RT-PCR, RPAs do not require extensive optimization and can be used for relative or absolute quantitation experiments without worrying about amplification-induced errors.
But RPAs can be time-consuming, and most scientists use radiolabeled probes, which are both hazardous and short-lived....
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