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Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue sections without protease treatment

Nature Protocols volume 5, pages 1061–1073 (2010)Cite this article

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Abstract

Noncoding RNAs (ncRNAs) comprise a diverse group of RNAs that function in essential cellular processes such as pre-mRNA splicing and mRNA translation and also regulate various aspects of gene expression in physiology and development. Methods of subcellular and tissue localization of ncRNAs are essential to understand their biological roles and their contribution to disease. We describe a rapid fluorescent (FISH) or chromogenic (CISH) in situ hybridization protocol for localization of ncRNAs (including microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), piwi-associated RNAs (piRNAs) and ribosomal RNAs (rRNAs)) in formalin-fixed, paraffin-embedded (FFPE) tissues and cultured cells, using locked nucleic acid (LNA)-modified oligonucleotides. In this protocol, sections are heated in citrate buffer, which eliminates the need for protease treatment, thus preserving optimal morphology and protein epitopes, and allowing the simultaneous detection of proteins with immunofluorescence staining (IF). LNA–FISH requires 5 h, or between 10 and 36 h when combined with IF; LNA–CISH requires 2 d.

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Figure 1
Figure 2: Codetection of 18S rRNA and Sm proteins in HeLa cells and FFPE mouse tissues using directly labeled LNA probes for FISH, combined with IF.
Figure 3: Codetection of miR-124 and neuron-specific class III b-tubulin (TuJ-1) in FFPE mouse brain by LNA–FISH with TSA amplification and IF, respectively.
Figure 4: Representative FISH micrographs of different ncRNAs in mouse FFPE tissues using directly labeled LNA probes.
Figure 5: LNA–CISH for piRNA (XL-piR-3) in Xenopus laevis oocyte.

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References

  1. Gesteland, R.F., Cech, T.R. & Atkins, J.F. The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2005).

  2. Liu, X., Fortin, K. & Mourelatos, Z. MicroRNAs: biogenesis and molecular functions. Brain Pathol. 18, 113–121 (2008).

    Article  CAS  Google Scholar 

  3. Hartig, J.V., Tomari, Y. & Forstemann, K. piRNAs—the ancient hunters of genome invaders. Genes Dev. 21, 1707–1713 (2007).

    Article  CAS  Google Scholar 

  4. Kirino, Y. et al. Arginine methylation of Piwi proteins catalysed by dPRMT5 is required for Ago3 and Aub stability. Nat. Cell Biol. 11, 652–658 (2009).

    Article  CAS  Google Scholar 

  5. Calin, G.A. & Croce, C.M. MicroRNA signatures in human cancers. Nat. Rev. Cancer 6, 857–866 (2006).

    Article  CAS  Google Scholar 

  6. Nicoloso, M.S. & Calin, G.A. MicroRNA involvement in brain tumors: from bench to bedside. Brain Pathol. 18, 122–129 (2008).

    Article  CAS  Google Scholar 

  7. Nelson, P.T., Wang, W.X. & Rajeev, B.W. MicroRNAs (miRNAs) in neurodegenerative diseases. Brain Pathol. 18, 130–138 (2008).

    Article  CAS  Google Scholar 

  8. Pardue, M.L. & Gall, J.G. Molecular hybridization of radioactive DNA to the DNA of cytological preparations. Proc. Natl. Acad. Sci. USA 64, 600–604 (1969).

    Article  CAS  Google Scholar 

  9. Baldino, F. Jr, Chesselet, M.F. & Lewis, M.E. High-resolution in situ hybridization histochemistry. Methods Enzymol. 168, 761–777 (1989).

    Article  CAS  Google Scholar 

  10. Zaidi, A.U., Enomoto, H., Milbrandt, J. & Roth, K.A. Dual fluorescent in situ hybridization and immunohistochemical detection with tyramide signal amplification. J. Histochem. Cytochem. 48, 1369–1375 (2000).

    Article  CAS  Google Scholar 

  11. Singer, R.H. & Ward, D.C. Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog. Proc. Natl. Acad. Sci. USA 79, 7331–7335 (1982).

    Article  CAS  Google Scholar 

  12. Farquharson, M., Harvie, R. & McNicol, A.M. Detection of messenger RNA using a digoxigenin end labelled oligodeoxynucleotide probe. J. Clin. Pathol. 43, 424–428 (1990).

    Article  CAS  Google Scholar 

  13. Morris, R.G. et al. Sensitivity of digoxigenin and biotin labelled probes for detection of human papillomavirus by in situ hybridisation. J. Clin. Pathol. 43, 800–805 (1990).

    Article  CAS  Google Scholar 

  14. van Gijlswijk, R.P. et al. Horseradish peroxidase-labeled oligonucleotides and fluorescent tyramides for rapid detection of chromosome-specific repeat sequences. Cytogenet. Cell Genet. 75, 258–262 (1996).

    Article  CAS  Google Scholar 

  15. Macechko, P.T., Krueger, L., Hirsch, B. & Erlandsen, S.L. Comparison of immunologic amplification vs enzymatic deposition of fluorochrome-conjugated tyramide as detection systems for FISH. J. Histochem. Cytochem. 45, 359–363 (1997).

    Article  CAS  Google Scholar 

  16. Speel, E.J., Saremaslani, P., Roth, J., Hopman, A.H. & Komminoth, P. Improved mRNA in situ hybridization on formaldehyde-fixed and paraffin-embedded tissue using signal amplification with different haptenized tyramides. Histochem. Cell Biol. 110, 571–577 (1998).

    Article  CAS  Google Scholar 

  17. Yang, H., Wanner, I.B., Roper, S.D. & Chaudhari, N. An optimized method for in situ hybridization with signal amplification that allows the detection of rare mRNAs. J. Histochem. Cytochem. 47, 431–446 (1999).

    Article  CAS  Google Scholar 

  18. Vester, B. & Wengel, J. LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43, 13233–13241 (2004).

    Article  CAS  Google Scholar 

  19. Wienholds, E. et al. MicroRNA expression in zebrafish embryonic development. Science 309, 310–311 (2005).

    Article  CAS  Google Scholar 

  20. Nelson, P.T. et al. RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain. RNA 12, 187–191 (2006).

    Article  CAS  Google Scholar 

  21. Silahtaroglu, A.N. et al. 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 (2007).

    Article  CAS  Google Scholar 

  22. Obernosterer, G., Martinez, J. & Alenius, M. Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections. Nat. Protoc. 2, 1508–1514 (2007).

    Article  CAS  Google Scholar 

  23. Solovei, I. et al. Spatial preservation of nuclear chromatin architecture during three-dimensional fluorescence in situ hybridization (3D-FISH). Exp. Cell Res. 276, 10–23 (2002).

    Article  CAS  Google Scholar 

  24. Kimura, T. et al. Quantification of in situ hybridization signals in rat testes. J. Histochem. Cytochem. 52, 813–820 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to the members of our laboratory and to Francesco Lotti for stimulating discussions and to Gideon Dreyfuss for providing the Y12 antibody. Supported by NIH grants GM0720777 and NS056070 to Z.M.

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Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Division of Neuropathology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA

    Mariàngels de Planell-Saguer & Zissimos Mourelatos

  2. Department of Cell Biology and Ecology, Faculty of Biology, University of Santiago de Compostela, Santiago de Compostela, Spain

    Mariàngels de Planell-Saguer & María Celina Rodicio

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  1. Mariàngels de Planell-Saguer
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Contributions

M.d.P.-S. developed the protocols and performed all the experiments. M.d.P.-S., M.C.R. and Z.M. contributed to the design and analysis of experiments and wrote the paper.

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Correspondence to Zissimos Mourelatos.

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The authors declare no competing financial interests.

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de Planell-Saguer, M., Rodicio, M. & Mourelatos, Z. Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue sections without protease treatment. Nat Protoc 5, 1061–1073 (2010). https://doi.org/10.1038/nprot.2010.62

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