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Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification

Nature Protocols volume 2, pages 2520–2528 (2007)Cite this article

Abstract

The ability to determine spatial and temporal microRNA (miRNA) accumulation at the tissue, cell and subcellular levels is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. This protocol describes a method for fast and effective detection of miRNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA)-modified oligonucleotide probes with FISH using the tyramide signal amplification (TSA) technology. Although both approaches have previously been shown to increase detection sensitivity in FISH, combining these techniques into one protocol significantly decreases the time needed for miRNA detection in cryosections, while simultaneously retaining high detection sensitivity. Starting with fixation of the tissue sections, this miRNA FISH protocol can be completed within approximately 6 h and allows miRNA detection in a wide variety of animal tissue cryosections as well as in human tumor biopsies at high cellular resolution.

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Figure 1: Demonstration of the specificity of the locked nucleic acid (LNA)-fluorescence in situ hybridization (FISH) method for detection of microRNAs (miRNA).
Figure 2: In situ detection of specific microRNAs in mouse cryosections from different tissues.
Figure 3: Comparison of microRNA expression between different or within the same species.
Figure 4: Detection of tumor-specific accumulation of miR-21 in human bladder biopsies.
Figure 5: Flowchart of the microRNA in situ detection method, depicting the main steps of the miRNA fluorescence in situ hybridization (FISH) protocol.
Figure 6

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References

  1. Bartel, D.P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).

    Article  CAS  Google Scholar 

  2. Wienholds, E. & Plasterk, R.H. MicroRNA function in animal development. FEBS Lett. 579, 5911–5922 (2005).

    Article  CAS  Google Scholar 

  3. Kloosterman, W.P. & Plasterk, R.H. The diverse functions of microRNAs in animal development and disease. Dev. Cell. 11, 441–450 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  4. Brennecke, J., Hipfner, D.R., Stark, A., Russel, R.B. & Cohen, S.M. Bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 113, 25–36 (2003).

    Article  CAS  Google Scholar 

  5. Mansfield, J.H. et al. MicroRNA-responsive 'sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression. Nat. Genet. 36, 1079–1083 (2004).

    Article  CAS  PubMed Central  Google Scholar 

  6. Koshkin, A.A. et al. 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 (1998).

    Article  CAS  Google Scholar 

  7. Braasch, D.A. & Corey, D.R. Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA. Chem. Biol. 8, 1–7 (2001).

    Article  CAS  PubMed Central  Google Scholar 

  8. Kurreck, J., Wyszko, E., Gillen, C. & Erdmann, V.A. Design of antisense oligonucleotides stabilized by locked nucleic acids. Nucleic Acids Res. 30, 1911–1918 (2002).

    Article  CAS  PubMed Central  Google Scholar 

  9. Jacobsen, N. et al. Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture. Nucleic Acids Res. 32, e64 (2004).

    Article  PubMed Central  Google Scholar 

  10. Kauppinen, S., Vester, B. & Wengel, J. Locked nucleic acid: high-affinity targeting of complementary RNA for RNomics. Handb. Exp. Pharmacol. 173, 405–422 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Ason, B. et al. Differences in vertebrate microRNA expression. Proc. Natl. Acad. Sci. USA. 103, 14385–14389 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  13. Darnell, D.K. et al. MicroRNA expression during chick embryo development. Dev. Dyn. 235, 3156–3165 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  14. Kloosterman, W.P., Wienholds, E., de Bruijn, E., Kauppinen, S. & Plasterk, R.H. In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat. Methods 3, 27–29 (2006).

    Article  CAS  Google Scholar 

  15. Wheeler, G., Ntounia-Fousara, S., Granda, B., Rathjen, T. & Dalmay, T. Identification of new central nervous system specific mouse microRNAs. FEBS Lett. 580, 2195–2200 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  16. 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  PubMed Central  Google Scholar 

  17. Obernosterer, G., Leuschner, P.J., Alenius, M. & Martinez, J. Post-transcriptional regulation of microRNA expression. RNA 12, 1161–1167 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  18. 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  PubMed Central  Google Scholar 

  19. Silahtaroglu, A.N., Tommerup, N. & Vissing, H. FISHing with locked nucleic acids (LNA): evaluation of different LNA/DNA mixmers. Mol. Cell. Probes 17, 165–169 (2003).

    Article  CAS  PubMed Central  Google Scholar 

  20. Silahtaroglu, A., Pfundheller, H., Koshkin, A., Tommerup, N. & Kauppinen, S. LNA-modified oligonucleotides are highly efficient as FISH probes. Cytogenet. Genome Res. 107, 32–37 (2004).

    Article  CAS  PubMed Central  Google Scholar 

  21. Christoffersen, N.R., Silahtaroglu, A., Orom, U.A., Kauppinen, S. & Lund, A.H. miR-200b mediates post-transcriptional repression of ZFHX1B. RNA 13, 1172–1178 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  22. Kerstens, H.M., Poddighe, P.J. & Hanselaar, A.G. A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramide. J. Histochem. Cytochem. 43, 347–352 (1995).

    Article  CAS  PubMed Central  Google Scholar 

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Acknowledgements

The authors wish to thank Anna Catharina Lassen, Ha Nguyen, Ursula Rentzmann, Lene Løgstrup and Eniser Zekirovska for their excellent technical assistance. This study was supported by grants from the Danish National Advanced Technology Foundation, Danish Medical Research Council to S.K., Danish Research Agency to A.N.S. and the Lundbeck Foundation to S.K. and A.N.S. Wilhelm Johannsen Centre for Functional Genome Research is established by the Danish National Research Foundation.

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Authors and Affiliations

  1. Department of Cellular and Molecular Medicine, Wilhelm Johannsen Centre for Functional Genome Research, University of Copenhagen, Blegdamsvej 3, Copenhagen N, DK-2200, Denmark

    Asli N Silahtaroglu, Niels Tommerup & Sakari Kauppinen

  2. Department of Orthodontics, School of Dentistry, University of Copenhagen, Blegdamsvej 3, Copenhagen N, DK-2200, Denmark

    Dorrit Nolting

  3. Department of Clinical Biochemistry, Molecular Diagnostic Laboratory, Aarhus University Hospital, Skejby, DK-8200, Aarhus N, Denmark

    Lars Dyrskjøt

  4. Hubrecht Institute, Uppsalalaan 8, Utrecht, 3584CT, The Netherlands

    Eugene Berezikov

  5. Department of Neuroscience and Pharmacology, University of Copenhagen, Blegdamsvej 3, Copenhagen N, DK-2200, Denmark

    Morten Møller

  6. Santaris Pharma, Boege Alle 3, DK-2970, Denmark

    Sakari Kauppinen

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  1. Asli N Silahtaroglu
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Correspondence to Asli N Silahtaroglu.

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Silahtaroglu, A., Nolting, D., Dyrskjøt, L. 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). https://doi.org/10.1038/nprot.2007.313

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