RNase+protection

=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