Northern blotting enables the specific detection and characterization of RNA molecules. Recently, circular RNAs (circRNAs) were described as a new class of cell type-specific noncoding RNAs. With the discovery of many novel circRNAs on the basis of high-throughput sequencing and bioinformatics, a solid biochemical approach is required to directly detect and validate specific circRNA species. Here we give a detailed overview of how different Northern blot methods can be employed to validate specific circRNAs. Different Northern gel and detection systems are introduced, in combination with additional tools for circRNA characterization, such as RNase R and RNase H treatments.
Keywords: Circular RNA; Denaturing glyoxal agarose gel; Digoxigenin-detection system; Junction probes; Northern blot; Northern transfer; RNase H; RNase R; Riboprobes; circRNA.