Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs
Blower, Mike D.
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CitationSharp, Judith A., and Mike D. Blower. 2013. “Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs.” Journal of Visualized Experiments : JoVE (76): 50434. doi:10.3791/50434. http://dx.doi.org/10.3791/50434.
AbstractMany organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:11855915
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