Splinted Ligation Method

Abstract

The Splinted-ligation technology was initially developed by Moore and Query (Methods Enzymol 317:109–123, 2000; Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, School of Medicine, Ohio, USA) for joining two or more RNA fragments by including bridging splint DNA templates to create RNA:RNA/DNA complexes which are nicked specifically at the desired site of ligation substrates of the T4 DNA ligase. This strategy was later adapted by Maroney et al. (Nat Struct Mol Biol 13:1102–1107, 2006, RNA 13:930–936, 2007 and Nat Protoc 3:279–287, 2008) for detecting miRNA expression. The Splinted-ligation technology is a protocol for the direct labeling and quantitative detection of small RNAs, based on nucleic acid hybridization technology for characterizing small RNAs of known sequence, such as mature miRNAs. Compared with Northern blotting, the Splinted Ligation technique possesses the advantage of simplicity: it takes advantage of liquid hybridization kinetics and avoids the transfer, prehybridization, and washing steps required for Northern blotting. It also allows easy processing of multiple samples. The Splinted Ligation technology is ~50 times more sensitive than Northern blotting using DNA probes (RNA 13:930–936, 2007). The technology is convenient and straightforward, and does not require specialized, sophisticated equipment or any amplification step. The Splinted-ligation technology has the same applications as Northern blot and many other methods such as expression analysis and validation of microarray studies. It has been successfully used to detect and quantify different classes of small RNAs in unfractionated RNA samples from nanogram to microgram amounts of total RNA without an amplification step.