Direct miRNA detection using splinted ligation

Abstract

We have developed a sensitive and specific detection assay for small RNAs based on splinted-ligation technique. Presently, Northern blot is the standard method for miRNA detection. However, major drawbacks of Northern blots are time consuming procedures and poor sensitivity, especially when monitoring expression of short nucleotide sequences such as miRNAs. Here, we describe a new assay for direct labeling and detection of mature miRNA from total RNA by splinted-ligation technique using miRtect-IT™ miRNA Labeling and Detection kit (USB). The splinted-ligation technique is a nucleic acid hybridization assay that uses a miRNA-specific bridge oligonucleotide to form base pairs with the miRNA and a detection oligonucleotide. The captured miRNA is subsequently ligated to the detection oligonucleotide to generate labeled miRNAs. A comparison with Northern blot data showed a 50-fold increase in detection sensitivity. The assay has a linear detection range of 0.1–10 femtomoles and can be completed within 6 hours. We also demonstrated that this method can be used to directly label and detect miRNAs from a variety of biological samples. The results obtained using the miRtect-IT™ technology are in agreement with published studies that have characterized organ-specific miRNA expression in animals, regulation of plant miRNAs in flower and seedlings, and testis-specific expression of piRNAs.