title: Production of Xenopus tropicalis egg extracts to identify microtubule-associated RNAs.
abstract: Many 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.
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, MA, USA.
authors.forename: Judith A
authors.initials: JA
authors.lastname: Sharp
authors.forename: Mike D
authors.initials: MD
authors.lastname: Blower
mesh_terms: D000818:Animals; D002457:Cell Extracts / Q000737:chemistry*; D005260:Female; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism; D010063:Ovum / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014981:Xenopus
chemical_list: D002457:Cell Extracts; D012333:RNA, Messenger