In Situ Hybridization for Fungal Ribosomal RNA Sequences in Paraffin-Embedded Tissues Using Biotin-Labeled Locked Nucleic Acid Probes

  • Kathleen T. MontoneEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1211)


Ribosomal RNAs (rRNA) are conserved, abundant species-specific sequences that are used for phylogenetically classifying organisms. Due to their abundance and species specificity, rRNA sequences have been established as optimal targets for in situ hybridization (ISH). ISH for rRNA sequences using DNA oligonucleotide probes has been utilized to detect a variety of fungi in paraffin tissues. However, ISH with some oligonucleotide DNA probes produces weak signals, and applications for nucleotide modification may be useful to enhance hybridization signal. ISH with LNA probes has been shown to result in improved ISH signal. A protocol for LNA ISH with biotin-labeled LNA oligonucleotide probes is described.

Key words

In situ hybridization Locked nucleic acids LNA Fungi Aspergillus rRNA 


  1. 1.
    Olsen GJ, Woese CR (1993) Ribosomal RNA: a key to phylogeny. FASEB J 7(1):113–123PubMedGoogle Scholar
  2. 2.
    Olsen GJ, Woese CR, Overbeek R (1994) The winds of (evolutionary) change: breathing new life into microbiology. J Bacteriol 176(1):1–6PubMedPubMedCentralGoogle Scholar
  3. 3.
    Montone KT (1994) In situ hybridization for ribosomal RNA sequences: a rapid sensitive method for diagnosis of infectious pathogens in anatomic pathology substrates. Acta Histochem Cytochem 27:601–606CrossRefGoogle Scholar
  4. 4.
    Montone KT, Litzky LA (1995) Rapid method for detection of Aspergillus 5S ribosomal RNA using a genus-specific oligonucleotide probe. Am J Clin Pathol 103:48–51PubMedGoogle Scholar
  5. 5.
    Hayden RT, Isotalo PA, Parrett T et al (2003) In situ hybridization for the differentiation of Aspergillus, Fusarium, and Pseudallescheria species in tissue section. Diagn Mol Pathol 12(1):21–26PubMedCrossRefGoogle Scholar
  6. 6.
    Hayden RT, Qian X, Procop GW et al (2002) In situ hybridization for the identification of filamentous fungi in tissue section. Diagn Mol Pathol 11(2):119–126PubMedCrossRefGoogle Scholar
  7. 7.
    Hayden RT, Qian X, Roberts GD et al (2001) In situ hybridization for the identification of yeastlike organisms in tissue section. Diagn Mol Pathol 10(1):15–23PubMedCrossRefGoogle Scholar
  8. 8.
    Kaur H, Wengel J, Maiti S (2008) Thermodynamics of DNA-RNA heteroduplex formation: effects of locked nucleic acid nucleotides incorporated into the DNA strand. Biochemistry 47:1218–1227PubMedCrossRefGoogle Scholar
  9. 9.
    Kurreck J, Wyszko E, Gillen C et al (2002) Design of antisense oligonucleotides stabilized by locked nucleic acids. Nucleic Acids Res 30:1911–1918PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Koshkin AA, Nielsen P, Meldgaard M et al (1998) LNA (locked nucleic acid): an RNA mimic forming exceedingly stable LNA:LNA duplexes. J Am Chem Soc 120:13252–13253CrossRefGoogle Scholar
  11. 11.
    Amann R, Fuchs BM (2008) Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat Rev Microbiol 6(5):339–348PubMedCrossRefGoogle Scholar
  12. 12.
    Thomsen R, Nielsen PS, Jensen TH (2005) Dramatically improved RNA in situ hybridization signals using LNA-modified probes. RNA 11:1745–1748PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Kubota K, Ohashi A, Imachi H et al (2006) Improved in situ hybridization efficiency with locked-nucleic-acid-incorporated DNA probes. Appl Environ Microbiol 72(8):5311–5317PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Montone KT (2008) In situ hybridization for fungal pathogens using locked nucleic acid probes. FASEB J 22(708.4)Google Scholar
  15. 15.
    Montone KT, Feldman MD (2009) In situ detection of Aspergillus ribosomal rRNA sequences using a locked nucleic acid (LNA) probe. Diagn Mol Pathol 18(4):239–242PubMedCrossRefGoogle Scholar
  16. 16.
    Montone KT, Feldman MD, Peterman H et al (2010) In situ hybridization for Coccidioides immitis 5.8S ribosomal RNA sequences in formalin-fixed, paraffin- embedded pulmonary nodules using a locked nucleic acid (LNA) probe: a rapid means for speciation in tissue sections. Diagn Mol Pathol 19(2):99–104PubMedCrossRefGoogle Scholar
  17. 17.
    Baliff J, Litzky L, Montone KT (2010) Rapid in situ hybridization for fungal pathogens in lung specimens using locked nucleic acid probes. Arch Pathol Lab Med 134(2):229–234Google Scholar
  18. 18.
    Montone KT (2009) Differentiation of Fusarium from Aspergillus species by colorimetric in situ hybridization in formalin-fixed, paraffin-embedded tissue sections using dual fluorogenic-labeled LNA probes. Am J Clin Pathol 132(6):866–870PubMedCrossRefGoogle Scholar
  19. 19.
    Montone KT, Feldman MD (2009) Evaluation of DNA, LNA, and PNA probes for in situ detection of panfungal ribosomal RNA sequences in tissues: a multispectral imaging study. Modern Pathol 22(Suppl 1)Google Scholar
  20. 20.
    Kieran JA Strategies for preventing detachment of sections from glass slides.
  21. 21.
    Iezzoni JC, Kang JH, Montone KT et al (1992) Colorimetric detection of herpes simplex virus by DNA in situ sandwich hybridization: a rapid, formamide-free, random oligomer-enhanced method. Nucleic Acids Res 20(5):1149–1150PubMedCrossRefPubMedCentralGoogle Scholar
  22. 22.
    Park CS, Manahan LJ, Brigati DJ (1991) Automated molecular pathology: one hour in situ DNA hybridization. J Histotechnol 14: 219–229CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Pathology and Laboratory Medicine, Perelman School of MedicineHospital of the University of PennsylvaniaPhiladelphiaUSA

Personalised recommendations