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.
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Carlile, M., Werner, A. (2014). Extraction and Nonradioactive Detection of Small RNA Molecules. In: Werner, A. (eds) Animal Endo-SiRNAs. Methods in Molecular Biology, vol 1173. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0931-5_8
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DOI: https://doi.org/10.1007/978-1-4939-0931-5_8
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0930-8
Online ISBN: 978-1-4939-0931-5
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