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Extraction and Nonradioactive Detection of Small RNA Molecules

  • Mark CarlileEmail author
  • Andreas Werner
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1173)

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.

Keywords

siRNA Nonradioactive probe Digoxigenin Northern blotting endo-siRNA 

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Faculty of Applied SciencesUniversity of SunderlandSunderlandUK
  2. 2.RNA Biology Group, Institute for Cell and Molecular BiosciencesNewcastle UniversityNewcastleUK

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