MicroRNA detection by northern blotting using locked nucleic acid probes

MicroRNAs (miRNAs) are short, about 21 nucleotides in length, noncoding, regulatory RNA molecules representing a new layer in post-transcriptional regulation of gene expression. Intensive miRNA research has necessitated the development of effective miRNA detection methods such as northern analyses, quantitative real-time PCR and microarrays. Northern analysis is a widely used method for miRNA analyses because it is generally a readily available technology for laboratories and does not require special equipment and technical knowledge. The major disadvantages of the northern blot technology using the traditional DNA oligonucleotide probes are its poor sensitivity and the high time consumption. Here, we describe an improved protocol for miRNA northern blot analysis, which includes RNA extraction, polyacrylamide gel electrophoresis and northern blotting, and the hybridization and detection of locked nucleic acid (LNA)-modified oligonucleotide probes. The use of LNA-modified oligonucleotide probes allows highly sensitive and specific detection of mature miRNAs and also dramatically reduces the period of time necessary for carrying out the protocol. Using this approach, the hybridization, washing and signal-detection steps can be performed ideally in 4 h.