TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos
TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos
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DOI：
10.3791/52985
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发表时间：
2015-09-01
影响因子：
1.2
通讯作者：
Junion, Guillaume
中科院分区：
文献类型：
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作者：
Bertin, Benjamin;Renaud, Yoan;Junion, Guillaume
测量单个细胞中翻译过程中mRNA的水平提供了在给定时间点参与细胞功能的蛋白质的信息。被称为翻译核糖体亲和纯化（TRAP）的协议能够以细胞类型特异性的方式捕获这种mRNA翻译过程。基于携带标记的核糖体亚基的多核糖体的亲和纯化，TRAP可以应用于单个细胞中的翻译组分析，使得在发育过程中比较细胞类型或在分子水平上跟踪疾病发展进程和潜在疗法的影响成为可能。在这里，我们报告了TRAP协议的优化版本，称为TRAP-rc（稀有细胞），致力于从稀有细胞群体中识别翻译中的RNA。TRAP-rc使用Gal 4/UAS靶向系统在果蝇胚胎中的肌细胞的有限群体中进行验证。这种新的方案允许回收足够量的细胞类型特异性RNA，用于全球基因表达分析，如微阵列或RNA-seq。该方案的鲁棒性和Gal 4驱动程序的大量收集使得TRAP-rc成为一种高度通用的方法，在细胞特异性全基因组研究中具有潜在的应用。
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 genomewide studies.