title: Extraction and nonradioactive detection of small RNA molecules.
abstract: The emergence of small RNAs as key and potent regulators of gene expression has prompted the need for robust detection and assay protocols to be developed for investigating their generation and tissue distribution. The physicochemical nature of these RNAs allows traditional assay methods to be employed; however, due to the relatively small size of endo-siRNAs, key changes to these protocols are required. Here, we present a method for the nonradioactive detection of endo-siRNAs in mouse tissue and microinjected Xenopus oocytes. The method comprises steps for RNA extraction, PAGE, and low-stringency northern blotting using DIG-labelled RNA probes. Moreover, it includes a strategy to design and generate cheap hybridization probes with greatly increased sensitivity. These methods may be used as a simple and robust protocol for nonradioactive detection of small RNAs or be combined with other strategies to potentially enhance signal intensity.
authors.affiliation: Faculty of Applied Sciences, University of Sunderland, Wharncliffe Street, Sunderland, SR1 3SD, UK, Mark.Carlile@sunderland.ac.uk.
authors.forename: Mark
authors.initials: M
authors.lastname: Carlile
authors.forename: Andreas
authors.initials: A
authors.lastname: Werner
mesh_terms: D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D051379:Mice; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009865:Oocytes / Q000737:chemistry; D015347:RNA Probes / Q000737:chemistry; D034741:RNA, Small Interfering / Q000032:analysis* / Q000302:isolation & purification; D014981:Xenopus
chemical_list: D015347:RNA Probes; D034741:RNA, Small Interfering