RNA Whole‐Mount In Situ Hybridization Proximity Ligation Assay (rISH‐PLA), an Assay for Detecting RNA‐Protein Complexes in Intact Cells

Ioannis M. Roussis1, Fiona A. Myers1, Garry P. Scarlett1

1 Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.20
DOI:  10.1002/cpcb.13
Online Posting Date:  March, 2017
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Techniques for studying RNA‐protein interactions have lagged behind those for DNA‐protein interactions as a consequence of the complexities associated with working with RNA. This unit describes a method for the adaptation of the In Situ Hybridization—Proximity Ligation Assay (ISH‐PLA) to the study of RNA regulation (rISH‐PLA). 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 heterogeneous 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. © 2017 by John Wiley & Sons, Inc.

Keywords: ISH‐PLA; protein‐nucleic acid interactions; RNA

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: rISH‐PLA Standard Protocol
  • Support Protocol 1: RNA Structure Determination and Probe Design
  • Support Protocol 2: Mounting the Sample
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: rISH‐PLA Standard Protocol

  • Sample: whole Xenopus oocytes or embryos
  • 4% paraformaldehyde in phosphate‐buffered saline (PBS; Sigma‐Aldrich, cat. no. P4417); store at −20°C
  • Methanol (Sigma‐Aldrich); store at room temperature
  • PBST: 0.02% (v/v) Tween 20 in phosphate‐buffered saline (PBS; Sigma‐Aldrich, cat. no. P4417)
  • Triethanolamine (Sigma‐Aldrich); store at room temperature
  • Acetic anhydride (Sigma‐Aldrich); store at room temperature
  • Hybridization buffer (see recipe)
  • LNA probe (synthesized by Exiqon; see protocol 2
  • Deionized formamide (Sigma‐Aldrich); store at room temperature
  • 20× saline sodium citrate (SSC; Sigma‐Aldrich); store at room temperature
  • Tween 20 (Sigma‐Aldrich; store at room temperature)
  • Blocking Reagent (Sigma‐Aldrich, cat. no. 110961760)
  • Maleic Acid Block (MAB; see recipe)
  • Duolink In Situ Red Starter Kit Mouse/Rabbit (Sigma‐Aldrich); store at 4°C.
  • Anti‐biotin primary antibody: typically mouse (Abcam); store at −20°C
  • Anti‐target secondary antibody: typically rabbit anti–mouse IgG (either user‐raised or purchased from Abcam); store at 4°C
  • Sigma water
  • Heat block
  • Rocker
  • 250‐μl PCR tubes
  • Confocal microscope

Support Protocol 1: RNA Structure Determination and Probe Design

  • Transparent nail polish
  • 100% glycerol as mounting medium
  • Sample: oocytes or embryos prepared for rISH‐PLA (Basic Protocol 1, step 25)
  • Microscope slides and coverslips
  • Glass cutter
  • Fine tweezers (optional)
  • Confocal microscope
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Literature Cited

  Braasch, D. and Corey, D.R. 2001. Locked nucleic acid (LNA): Fine‐tuning the recognition of DNA and RNA. Chem. Biol. 8:1‐7. doi: 10.1016/S1074‐5521(00)00058‐2.
  Goljanek‐Whysall, K., Mok, G.F., Alrefaei, A.F., Kennerley, N., Wheeler, G.N., and Münsterberg, A. 2014. MyomiR‐dependent switching of BAF60 variant incorporation into Brg1 chromatin remodeling complexes during embryo myogenesis. Development 2:3378‐3387. doi: 10.1242/dev.108787.
  Gomez, D., Shankman, L.S., Nguyen, T., and Owens, G.K. 2013. Detection of histone modifications at specific gene loci in single cells in histological sections. Nat. Methods 10:171‐177. doi: 10.1038/nmeth.2332.
  Koos, B., Andersson, L., Clausson, C., Grannas, K., Klaesson, A., and Cane, G. 2014. Analysis of protein interactions in situ by proximity ligation assays. Curr. Top. Microbiol. Immunol. 377:111‐126. doi: 10.1007/82_2013_334.
  Koshkin, A., Singh, S.K., Nielsen, P., Rajwanshi, V.K., Kumar, R., and Meldgaard, M. 1998. LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5‐methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron 54:3607‐3630. doi: 10.1016/S0040‐4020(98)00094‐5.
  Niranjanakumari, S., Lasda, E., Brazas, R., and Garcia‐Blanco, M. 2002. Reversible cross‐linking combined with immunoprecipitation to study RNA‐protein interactions in vivo. Methods 26:182‐190. doi: 10.1016/S1046‐2023(02)00021‐X.
  Obernosterer, G., Martinez, J., and Alenius, M. 2007. Locked nucleic acid‐based in situ detection of microRNAs in mouse tissue sections. Nat. Protoc. 2:1508‐1514. doi: 10.1038/nprot.2007.153.
  Roussis, I.M., Guille, M., Myers, F.A., and Scarlett, G.P. 2016. RNA Whole‐mount in situ hybridisation proximity ligation assay (rISH‐PLA), an assay for detecting RNA‐protein complexes in intact cells. PLoS One 11:e0147967. doi: 10.1371/journal.pone.0147967.
  Shankman, L.S., Gomez, D., Cherepanova, O.A., Salmon, M., Alencar, G.F., and Haskins, R.M. 2015. KLF4‐dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis. Nat. Med. 21:628‐637. doi: 10.1038/nm.3866.
  Silahtaroglu, A.N., Nolting, D., Dyrskjøt, L., Berezikov, E., Møller, M., and Tommerup, N. 2007. Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification. Nat. Protoc. 2:2520‐2528. doi: 10.1038/nprot.2007.313.
  Ule, J., Jensen, K., Mele, A., and Darnell, R.B. 2005. CLIP: A method for identifying protein‐RNA interaction sites in living cells. Methods 37:376‐386. doi: 10.1016/j.ymeth.2005.07.018.
  Vester, B. and Wengel, J. 2004. LNA (Locked Nucleic Acid): High‐affinity targeting of RNA and DNA. Biochemistry 43:13233‐13241. doi: 10.1021/bi0485732.
  Wang, Z., Tollervey, J., Briese, M., Turner, D., and Ule, J. 2009. CLIP: Construction of cDNA libraries for high‐throughput sequencing from RNAs cross‐linked to proteins in vivo. Methods 48:287‐293. doi: 10.1016/j.ymeth.2009.02.021.
Key References
  Gomez et al., 2013. See above.
  ISH‐PLA as applied to DNA and epigenetics.
  Roussis et al., 2016. See above.
  Our original paper detailing the rISH‐PLA protocol as applied to Xenopus oocytes.
Internet Resources
  UNAfold Web server.
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