In the RPA (Ribonuclease Protection Assay), what is the function of RNase A and T1?
Ribonuclease Protection Assay relies on solution hybridization between a labeled antisense RNA probe and target mRNA, forming a double-stranded hybrid resistant to single-strand-specific ribonucleases. RNase A, which cleaves after cytosine and uridine residues, and RNase T1, which cleaves after guanosine, are added to digest all unhybridized single-stranded RNA, including excess probe and non-target RNAs. The protected double-stranded fragment remains intact and is resolved on denaturing PAGE. Quantification of its intensity reflects abundance of specific transcript, allowing sensitive detection of low-level RNAs.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.