The major disadvantages of the northern blot technology using the traditional DNA oligonucleotide probes are its poor sensitivity and the high time consumption. 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. Intensive miRNA research has necessitated the development of effective miRNA detection methods such as northern analyses, quantitative real-time PCR and microarrays. Although chemical cross-linking takes longer (15 min to 2 h) than UV cross-linking, improved sensitivity means shorter periods of exposure are required to detect signal after hybridization.read more read lessĪbstract: MicroRNAs (miRNAs) are short, about 21 nucleotides in length, noncoding, regulatory RNA molecules representing a new layer in post-transcriptional regulation of gene expression. Northern blotting can be done in 2 d, but detection of a specific RNA can vary from minutes to days. This requires no specialized equipment, is relatively inexpensive and is technically straightforward. We have replaced conventional UV-cross-linking of RNA to nylon membranes with a novel, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated, chemical cross-linking step that enhances detection of small RNA by up to 50-fold. RNA of interest is then detected by hybridization with labeled complementary nucleic acid probes. Northern blot analysis involves the separation of RNA molecules by denaturing gel electrophoresis followed by transfer and cross-linking of the separated molecules to nylon membrane. Abstract: This protocol describes an improved northern blot method that enhances detection of small RNA molecules (<40 nt) including regulatory species such as microRNA (miRNA), short-interfering RNA (siRNA) and Piwi-interacting RNA.
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