HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2′ OH of the 3′-terminal nucleotide. A distinct small RNA pathway silences selfish genetic elements in the germline. RNA interference is mediated by 21- and 22-nucleotide RNAs. UV crosslinking of RNA to nylon membrane enhances hybridization signals. Application to specific modification of DNA. Photoreaction of thymidine with primary amines. MicroRNA maturation: stepwise processing and subcellular localization. The nuclear RNase III Drosha initiates microRNA processing. Real-time expression profiling of microRNA precursors in human cancer cell lines. 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. 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. 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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