Unbound, single-stranded RNA can be digested by RNase (A or T1) to ribonucleotides, whereas double-stranded RNA is not digested by RNase. Based on this principle, the RNase Protection Assay (RPA) is used to validate chimeric RNAs. Importantly, this assay does not employ reverse transcription (RT), thus avoiding potential false-positive results which could occur during RT such as template-switching. We first generate RNA probes with 32phosphate (P) or biotin that are complementary to the predicted nucleotide sequence of the chimeric RNA, then hybridize them to RNA samples. The labeled RNA probes can bind specifically with the target chimeric RNA in order to form double-stranded RNA. This newly formed RNA is resistant to digestion by RNase and therefore can be identified by high-resolution, denaturing polyacrylamide gel electrophoresis.
Keywords: Chimeric RNA; Denaturing polyacrylamide gel electrophoresis; Double-stranded RNA; RNase; Radioactively labeled; Single-stranded RNA.