Detection of Small Noncoding RNAs by In Situ Hybridization Using Probes of 2′-O-Methyl RNA + LNA

  • Martin Jensen Søe
  • Martin Dufva
  • Kim HolmstrømEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1173)


In situ hybridization is a powerful method to provide information about contextual distribution and cellular origin of nucleic acids, e.g., in formalin-fixed paraffin-embedded (FFPE) samples of tissue. Particularly the recently discovered classes of noncoding RNA (ncRNA) including endo-siRNAs and microRNAs require such a technique to enable their study and visualization in natural contexts, and in the last decade, many advances have been made, increasing our ability to specifically detect small ncRNAs. One of the key developments has been the demonstration of the superiority of using locked nucleic acid (LNA)-modified DNA probes for the detection of ncRNA in tissue. Here, we describe an alternative in situ hybridization protocol employing oligonucleotide probes consisting of combinations of LNA and 2´-O-methyl RNAs that under optimized hybridization buffer conditions can provide a highly sensitive assay performance with only 1 h hybridization time.


Noncoding RNA microRNA LNA In situ hybridization 2′-O-methyl RNA 



The authors thank the Danish Ministry and Agency of Science, Technology, and Innovation for funding to MJS.


  1. 1.
    Kloosterman WP, Wienholds E, de Bruijn E (2006) In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat Methods 3:27–29PubMedCrossRefGoogle Scholar
  2. 2.
    Silahtaroglu AN, Nolting D, Dyrskjøt L et al (2007) Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification. Nat Protoc 2:2520–2528PubMedCrossRefGoogle Scholar
  3. 3.
    Obernosterer G, Martinez J, Alenius M (2007) Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections. Nat Protoc 2:1508–1514PubMedCrossRefGoogle Scholar
  4. 4.
    Nuovo GJ, Elton TS, Nana-Sinkam P et al (2009) A methodology for the combined in situ analyses of the precursor and mature forms of microRNAs and correlation with their putative targets. Nat Protoc 4:107–115PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Pena JTG, Sohn-Lee C, Rouhanifard SH et al (2009) miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat Methods 6:139–141PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Jørgensen S, Baker A, Møller S et al (2010) Robust one-day in situ hybridization protocol for detection of microRNAs in paraffin samples using LNA probes. Methods 52:375–381PubMedCrossRefGoogle Scholar
  7. 7.
    Sempere LF, Preis M, Yezefski T et al (2010) Fluorescence-based codetection with protein markers reveals distinct cellular compartments for altered MicroRNA expression in solid tumors. Clin Cancer Res 16:4246–4255PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Nielsen BS (2012) MicroRNA in situ hybridization. Methods Mol Biol 822:67–84PubMedCrossRefGoogle Scholar
  9. 9.
    Renwick N, Cekan P, Masry PA et al (2013) Multicolor microRNA FISH effectively differentiates tumor types. J Clin Invest 123(6):2694–2702. doi:pii: 68760. 10.1172/JCI68760PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Koshkin AA, Singh SK, Nielsen P et al (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 54:3607–3630CrossRefGoogle Scholar
  11. 11.
    Vester B, Wengel J (2004) LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43:13233–13241PubMedCrossRefGoogle Scholar
  12. 12.
    McTigue PM, Peterson RJ, Kahn JD (2004) Sequence-dependent thermodynamic parameters for locked nucleic acid (LNA)-DNA duplex formation. Biochemistry 43:5388–5405PubMedCrossRefGoogle Scholar
  13. 13.
    Søe MJ, Møller T, Dufva M et al (2011) A sensitive alternative for microRNA in situ hybridizations using probes of 2′-O-methyl RNA + LNA. J Histochem Cytochem 59:661–672PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Nielsen BS, Holmstrøm K (2013) Combined microRNA in situ hybridization and immunohistochemical detection of protein markers. Methods Mol Biol 986:353–365PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Martin Jensen Søe
    • 1
  • Martin Dufva
    • 2
  • Kim Holmstrøm
    • 1
    Email author
  1. 1.Bioneer A/SHørsholmDenmark
  2. 2.DTU NanotechTechnical University of DenmarkLyngbyDenmark

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