title: Analysis of microRNA length variety generated by recombinant human Dicer.
abstract: miRNAs are a large subgroup of noncoding regulatory RNAs, which vary in length within the 20-25 nt range and show substantial length diversity and heterogeneity. To analyze the latter phenomenon, we recently developed high-resolution northern blotting and employed this method to investigate cleavages generated by recombinant human Dicer in the synthetic miRNA precursors. We paid special care to visualize clearly the cleavages generated by the individual RNase III domains of Dicer. We have compared the results of northern blotting with the results of standard analysis with the use of end-labeled RNA and visualization of Dicer cleavage products by autoradiography. The point-by-point steps of substrate preparation, recombinant Dicer cleavage assay, and northern blotting are described in this manuscript.
authors.affiliation: Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
authors.forename: Julia
authors.initials: J
authors.lastname: Starega-Roslan
authors.forename: Wlodzimierz J
authors.initials: WJ
authors.lastname: Krzyzosiak
mesh_terms: D015152:Blotting, Northern / Q000379:methods*; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D035683:MicroRNAs; D009841:Oligonucleotides; D011994:Recombinant Proteins; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases