RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA), an Assay for Detecting RNA-Protein Complexes in Intact Cells.
RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA), an Assay for Detecting RNA-Protein Complexes in Intact Cells.
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DOI：
10.1371/journal.pone.0147967
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Scarlett GP
中科院分区：
文献类型：
--
作者：
Roussis IM;Guille M;Myers FA;Scarlett GP
研究RNA-蛋白质相互作用的技术落后于研究dna -蛋白质复合物的技术，这是与RNA相关的复杂性的结果。在这里，我们提出了一种方法来修改现有的原位杂交-邻近连接试验（ISH-PLA）协议，使其适应RNA调控的研究（ISH-PLA）。作为原理证明，我们使用了非洲爪蟾Staufen RNA结合蛋白与Vg1 mRNA的相互作用，该复合物共定位于非洲爪蟾卵母细胞的植物极。识别Vg1 mRNA的Stau1抗体和锁定核酸探针（LNA）分别通过全挂免疫组织化学和全挂原位杂交实验独立验证了其适用性，然后将它们结合在rig - pla实验中。rig - pla检测允许在亚细胞和单细胞分辨率下识别给定的rna -蛋白复合物，从而避免了传统rna -蛋白相互作用检测技术在分析异质细胞群时缺乏空间分辨率和灵敏度的问题。这项技术对于研究复杂细胞混合物（例如组织切片或整个胚胎）中RNA结合蛋白（rbp）的活性特别有用。
Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein complexes as a consequence of the complexities associated with working with RNA. Here we present a method for the modification of the existing In Situ Hybridisation–Proximity Ligation Assay (ISH-PLA) protocol to adapt it to the study of RNA regulation (rISH-PLA). As proof of principle we used the well-characterised interaction of the Xenopus laevis Staufen RNA binding protein with Vg1 mRNA, the complex of which co-localises to the vegetal pole of Xenopus oocytes. The applicability of both the Stau1 antibody and the Locked Nucleic Acid probe (LNA) recognising Vg1 mRNA were independently validated by whole-mount Immunohistochemistry and whole-mount in situ hybridisation assays respectively prior to combining them in the rISH-PLA assay. The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogenous cell populations from which conventional RNA-protein interaction detection techniques suffer. This technique will be particularly usefully for studying the activity of RNA binding proteins (RBPs) in complex mixtures of cells, for example tissue sections or whole embryos.