The Detection of Hammerhead Ribozyme Cleavage by RT-PCR Methods

There are several methods available for the direct and indirect detection of hammerhead ribozyme-mediated cleavage of substrate RNAs in vivo. The indirect methods include the use of antisense and/or mutant ribozyme constructs to differentiate between inactivation by cleavage and inactivation by other means. In addition, comparison between a wild-type substrate and a mutant substrate, in which the target triplet has been altered to be a non-cleavable sequence, can provide indirect evidence for in vivo cleavage. The direct approaches (listed in increasing levels of sensitivity) include RNA analysis by Northern blot, RNase protection assays, and reverse transcriptase-PCR (RT-PCR) (but see also Chapters 33 and 34 ). To date there has only been one report of the direct analysis of cleavage products by Northern blot (1 ), and one group have successfully applied RNase protection assays (2 –4 ).