Skip to main content

In Situ Hybridization in Mineralized Tissues: The Added Value of LNA Probes for RNA Detection

  • Protocol
  • First Online:
Odontogenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1922))

  • 2956 Accesses

Abstract

In situ hybridization (ISH) is one of the fundamental methods in developmental biology and neurobiology. Their first ISH protocols were reported in 1969 (Gall and Pardue, Proc Natl AcadSci USA 63:378–83, 1969). Since several decades, ISH based on the specific hybridization of 100–2000 nucleotides long probes enabled the localization of DNA/RNA sequences in tissues and cells with high cellular resolution. But sometimes a limited sensitivity notably in mineralized tissues (Obernosterer et al., Nature Protocols 2:1508–14, 2007).

Here we describe a recent improvement of in situ hybridization efficiency by applying nucleotide locked nucleic acid (LNA)-incorporated oligodeoxynucleotide probes (20 LNA/DNA nucleotide probes) essentially used for noncoding miRNA and messenger RNAs.

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 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 219.99
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. Gu X, Li A, Liu S, Lin L, Xu S, Zhang P, Li S, Li X, Tian B, Zhu X, Wang X (2016) MicroRNA124 regulated neurite elongation by targeting OSBP. Mol Neurobiol 53(9):6388–6396

    Article  CAS  Google Scholar 

  2. Levsky JM, Singer RH (2003) Fluorescence in situ hybridization: past, present and future. J Cell Sci 116(Pt 14):2833–2838

    Article  CAS  Google Scholar 

  3. Stenvang J, Lindow M, Kauppinen S (2008) Targetings of microRNAs for therapeutics. Biochem Soc Trans 36(Pt 6):1197–1200

    Article  CAS  Google Scholar 

  4. Urbanek MO, Nawrocka AU, Krzyzosiak WJ (2015) Small RNA detection by in situ hybridization methods. Int J Mol Sci 16(6):13259–13286

    Article  CAS  Google Scholar 

  5. Koshkin AA, Singh SK, Nielsen P, Rajwanshi VK, Kumar R, Meldgaard M, Olsen CE, Wengel J (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

    Article  CAS  Google Scholar 

  6. Kumar R, Singh SK, Koshkin AA, Rajwanshi VK, Meldgaard M, Wengel J (1998) The first analogues of LNA (locked nucleic acids): Phosphorothioate-LNA and 2′-thio-LNA. Bioorg Med Chem Lett 8(16):2219–2222

    Article  CAS  Google Scholar 

  7. Obika S, Morio K, Hari Y, Imanishi T (1999) Preparation and properties of 2′, 5′-linked oligonucleotide analogues containing 3’-O,4’-C-methyleneribonucleosides. Bioorg Med Chem Lett 9(4):515–518

    Article  CAS  Google Scholar 

  8. Fritsch EF, Maniatis T (1989) Molecular cloning. In: Cold Spring Harbor Laboratory Press (ed) A laboratory manual, vol 3, 2nd edn. Cold Spring Harbor, New York, p 12

    Google Scholar 

  9. Cunningham CD 3rd, Schulte BA, Bianchi LM, Weber PC, Schmiedt BN (2001) Microwave decalcification of human temporal bones. Laryngoscope 111(2):278–282

    Article  Google Scholar 

  10. Jørgensen S, Baker A, Møller S, Nielsen BS (2010) Robust one-day in situ hybridization protocol for detection of microRNAs in paraffin samples using LNA probes. Methods 52(4):375–381

    Article  Google Scholar 

  11. Wilkinson DG, Nieto MA (1993) Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts. Methods Enzymol 225:361–373

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to D. Hotton .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Lignon, G., Hotton, D., Berdal, A., Bolaños, A. (2019). In Situ Hybridization in Mineralized Tissues: The Added Value of LNA Probes for RNA Detection. In: Papagerakis, P. (eds) Odontogenesis. Methods in Molecular Biology, vol 1922. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9012-2_18

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9012-2_18

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9011-5

  • Online ISBN: 978-1-4939-9012-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics