Skip to main content

LNA-FISH for Detection of MicroRNAs in Frozen Sections

  • Protocol
  • First Online:
Fluorescence in situ Hybridization (FISH)

Part of the book series: Methods in Molecular Biology ((MIMB,volume 659))

Abstract

MicroRNAs (miRNAs) are small (∼22 nt) noncoding RNA molecules that regulate the expression of protein coding genes either by cleavage or translational repression. miRNAs comprise one of the most abundant classes of gene regulatory molecules in multicellular organisms. Yet, the function of miRNAs at the tissue, cell, and subcellular levels is still to be explored. Especially, determining spatial and temporal expression of miRNAs has been a challenge due to their short size and low expression. This protocol describes a fast and effective method for detection of miRNAs in frozen tissue sections using fluorescence in situ hybridization. The method employs the unique recognition power of locked nucleic acids as probes together with enhanced detection power of the tyramide signal amplification system for detection of miRNAs in frozen tissues of human and animal origin within a single day.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Silahtaroglu AN, Nolting D, Dyrskjøt L, Berezikov E, Møller M, Tommerup N, Kauppinen S (2007). Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using Locked Nucleic Acid probes and tyramide signal amplification. Nat Protoc. 200, 2520–2528.

    Article  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  3. Kerstens HM, Poddighe PJ, Hanselaar AG (1995). A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramide. J Histochem Cytochem. 43, 347–352.

    Article  PubMed  CAS  Google Scholar 

  4. Thompson RC, Deo M, Turner DL (2007). Analysis of microRNA expression by in situ hybridization with RNA oligonucleotide probes. Methods. 43, 153–161.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  6. Kloosterman WP, Wienholds E, de Bruijn E, Kauppinen S, Plasterk RH (2006). In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat Methods. 3, 27–29.

    Article  PubMed  CAS  Google Scholar 

  7. Koshkin AA, Nielsen P, Meldgaard M, Rajwanshi VK, Singh SK, Wengel J (1998). LNA (locked nucleic acid): An RNA mimic forming exceedingly stable LNA: LNA duplexes. J Am Chem Soc. 120, 13252–13253.

    Article  CAS  Google Scholar 

  8. Stenvang J, Silahtaroglu AN, Lindow M, Elmen J, Kauppinen S (2008). The utility of LNA in microRNA-based cancer diagnostics and therapeutics. Semin Cancer Biol. 18, 89–102.

    Article  PubMed  CAS  Google Scholar 

  9. Schepeler T, Reinert JT, Ostenfeld MS, Christensen LL, Silahtaroglu AN, Dyrskjøt L, Wiuf C, Sørensen FJ, Kruhøffer M, Laurberg S, Kauppinen S, Ørntoft TF, Andersen CL (2008). Diagnostic and prognostic microRNAs in stage II colon cancer. Cancer Res. 68, 6416–6424.

    Article  PubMed  CAS  Google Scholar 

  10. Bak M, Silahtaroglu A, Møller M, Christensen M, Rath M, Skryabin B, Tommerup N, Kauppinen S (2008). MicroRNA expression in the adult mouse central nervous system. RNA. 14, 432–444.

    Article  PubMed  CAS  Google Scholar 

  11. Hébert SS, Horré K, Nicolaï L, Papadopoulou AS, Mandemakers W, Silahtaroglu A, Kauppinen S, Delacourte A, De Strooper B (2008). Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer’s disease correlates with increased BACE1/beta-secretase expression. Proc Natl Acad Sci U S A. 105, 6415–6420.

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

The author acknowledges the financial support from the Lundbeck Foundation, the Danish Research Agency and the Dr Sofus Carl Emil Friis and wife Olga Doris Friis Foundation. Wilhelm Johannsen Centre for Functional Genome Research is established by the Danish National Research Foundation.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Asli N. Silahtaroglu .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Silahtaroglu, A.N. (2010). LNA-FISH for Detection of MicroRNAs in Frozen Sections. In: Bridger, J., Volpi, E. (eds) Fluorescence in situ Hybridization (FISH). Methods in Molecular Biology, vol 659. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-789-1_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-789-1_11

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-788-4

  • Online ISBN: 978-1-60761-789-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics