title: TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos.
abstract: Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.
authors.affiliation: University of Clermont-Ferrand.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Bertin
authors.affiliation: University of Clermont-Ferrand.
authors.forename: Yoan
authors.initials: Y
authors.lastname: Renaud
authors.affiliation: Memorial Sloan Kettering Cancer Center.
authors.forename: Rajaguru
authors.initials: R
authors.lastname: Aradhya
authors.affiliation: University of Clermont-Ferrand; christophe.jagla@udamail.fr.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Jagla
authors.affiliation: University of Clermont-Ferrand.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Junion
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002846:Chromatography, Affinity / Q000379:methods; D004330:Drosophila; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods; D008280:Magnetics / Q000379:methods; D009132:Muscles / Q000737:chemistry / Q000166:cytology / Q000502:physiology; D011132:Polyribosomes / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics
chemical_list: D012333:RNA, Messenger