title: RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA), an Assay for Detecting RNA-Protein Complexes in Intact Cells.
abstract: 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.
authors.affiliation: Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, PO1 2DT, United Kingdom.
authors.forename: Ioannis M
authors.initials: IM
authors.lastname: Roussis
authors.affiliation: Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, PO1 2DT, United Kingdom.
authors.forename: Matthew
authors.initials: M
authors.lastname: Guille
authors.affiliation: Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, PO1 2DT, United Kingdom.
authors.forename: Fiona A
authors.initials: FA
authors.lastname: Myers
authors.affiliation: Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, PO1 2DT, United Kingdom.
authors.forename: Garry P
authors.initials: GP
authors.lastname: Scarlett
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001665:Binding Sites; D001681:Biological Assay; D001710:Biotin / Q000737:chemistry; D015870:Gene Expression; D007150:Immunohistochemistry / Q000379:methods*; D017403:In Situ Hybridization; D051379:Mice; D009841:Oligonucleotides / Q000737:chemistry; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D059010:Single-Cell Analysis / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D000906:Antibodies; C513103:GDF1 protein, Xenopus; D009841:Oligonucleotides; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C488764:STAU1 protein, Xenopus; D016212:Transforming Growth Factor beta; D029867:Xenopus Proteins; C477371:locked nucleic acid; D001710:Biotin
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