RNase A/T1 Protection Assay

RNase A/T₁ mapping provides a sensitive and quantitative method of expression analysis of known gene sequences. It differs from primer-derived analyses such as primer extension and reverse transcriptase-PCR by the use of a probe that is colinear with the transcript under study. This protocol has been applied to the analysis of plant RNA transcripts, for example, in the expression of endogenous plant genes (1 ), introduced genes (2 ), mutated or tagged genes (3 ), antisense gene copies (4 ), and in the analysis of plant nuclear pre-mRNA splicing (5 ). There are four steps involved in RNase A/T₁ analysis. First, the transcription of a labeled RNA probe complementary to the transcript under study. Second, hybridization of the labeled probe to the target RNA transcript. Third, digestion of the noncomplementary regions of the probe with ribonucleases. Finally, analysis of the digested products on denaturing polyacrylamide gels and autoradiography.