Analysis of Small RNAs by Northern Hybridization

This protocol describes how to use northern hybridization to detect 15- to 150-nt small RNAs. Total RNA is fractionated by electrophoresis through a denaturing polyacrylamide gel and then transferred to a nylon membrane by semidry electroblotting. After UV-cross-linking the RNA to the membrane, hybridization is performed in Church buffer using a ³²P-radiolabeled oligonucleotide probe followed by PhosphorImager analysis. The use of StarFire probes allows a substantial increase in the specific radioactivity of the hybridization probe. StarFire probes contain two domains. The target-specific domain at the 5' end hybridizes to the small RNA of interest. The universal template-binding domain at the 3' end is used to bind a universal template oligonucleotide that carries 10 deoxythymidines at its 5' end, providing a template for DNA polymerase to incorporate 10 [α-³²P]deoxyadenosines at the 3' end of the StarFire probe.