RNase protection


RNase protection is a method used in transcript analysis to map the start site of transcription.

The procedure is as follows:

1. Clone the gene of interest into a plasmid, downstream of a phage-specific promoter
2. Transcribe the gene using radio-labelled NTPs to produce an anti-sense RNA which is labelled throughout
3. Hybridise the radio-labelled anti-sense RNA with the gene
4. Digest the unhybridised (single-stranded) RNA with RNase A and RNase T1
5. Denature the protected, hybridised region and run in a polyacrylamide gel to quantify the label

RNase treatment is more effectively than the alternative S1 nuclease protection procedure for two reasons

1. RNA-RNA hybrids are more stable than RNA-DNA hybrids
2. More labelled nucleotides are present in the RNA, making RNase protection more sensitive than S1 nuclease protection