Improved northern blot detection of small RNAs using EDC crosslinking and DNA/LNA probes

Abstract

Successful detection of very small RNAs (tiny RNA, ~14 nt in length) by Northern blotting is dependent on improved Northern blot protocols that combine chemical crosslinking of RNA with 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide (EDC) to positively charged membranes, the use of native polyacrylamide gels, and the development of highly sensitive and specific probes modified with locked nucleic acids (LNA). In this protocol, we show that Northern blot detection of tiny RNAs with 5'-digoxigenin-labeled DNA/LNA mixmer probes is a highly sensitive and specific method and, in our hands, more sensitive than using a corresponding DNA/LNA mixmer probe with a 5'- 32 P-end label.