Chromogen Detection of microRNA in Frozen Clinical Tissue Samples Using LNA™ Probe Technology

  • Boye Schnack NielsenEmail author
  • Trine Møller
  • Kim Holmstrøm
Part of the Methods in Molecular Biology book series (MIMB, volume 1211)


Specific chromogen- and fluorescence-based detection of microRNA by in situ hybridization (ISH) in formalin-fixed and paraffin-embedded (FFPE) tissue sections has been facilitated by locked nucleic acid (LNA)-based probe technology and can be performed within a single working day. In the current method paper, we present a similar simple 1-day ISH method developed for cryostat sections obtained from clinical cryo-embedded tissue samples. The presented chromogen-based ISH method does not involve proteolytic pretreatment, which is mandatory for FFPE sections, but still retains a sensitivity level similar to that obtained in FFPE sections. The LNA-based ISH method is not only applicable in situations where only access to cryo-embedded material is possible, but it also has a potential use if combining microRNA ISH with immunohistochemistry in double fluorescence staining with antibodies not being compatible with proteolytic predigestion.

Key words

Chromogenic ISH Cryostat sections Frozen tissue In situ hybridization Locked nucleic acid microRNA 


  1. 1.
    Wienholds E, Kloosterman WP, Miska E et al (2005) MicroRNA expression in zebrafish embryonic development. Science 309:310–311PubMedCrossRefGoogle Scholar
  2. 2.
    Nielsen BS, Jorgensen S, Fog JU et al (2011) High levels of microRNA-21 in the stroma of colorectal cancers predict short disease-free survival in stage II colon cancer patients. Clin Exp Metastasis 28:27–38PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Sempere LF (2011) Integrating contextual miRNA and protein signatures for diagnostic and treatment decisions in cancer. Expert Rev Mol Diagn 11:813–827PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Quesne JL, Jones J, Warren J et al (2012) Biological and prognostic associations of miR-205 and let-7b in breast cancer revealed by in situ hybridization analysis of micro-RNA expression in arrays of archival tumour tissue. J Pathol 227:306–314PubMedCrossRefGoogle Scholar
  5. 5.
    Ason B, Darnell DK, Wittbrodt B et al (2006) Differences in vertebrate microRNA expression. Proc Natl Acad Sci U S A 103:14385–14389PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Yao X, Huang H, Xu L (2012) In situ detection of mature miRNAs in plants using LNA-modified DNA probes. In: Jin H, Gassmann W (eds) RNA abundance analysis, vol 883, Methods in molecular biology. Humana Press, New York, pp 143–154CrossRefGoogle Scholar
  7. 7.
    Memczak S, Jens M, Elefsinioti A et al (2013) Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495:333–338PubMedCrossRefGoogle Scholar
  8. 8.
    Qu Z, Adelson DL (2012) Identification and comparative analysis of ncRNAs in human, mouse and zebrafish indicate a conserved role in regulation of genes expressed in brain. PLoS One 7:e52275PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Neely LA, Patel S, Garver J et al (2006) A single-molecule method for the quantitation of microRNA gene expression. Nat Methods 3:41–46PubMedCrossRefGoogle Scholar
  10. 10.
    Sahu B, Sacui I, Rapireddy S et al (2011) Synthesis and characterization of conformationally preorganized, (R)-diethylene glycol-containing gamma-peptide nucleic acids with superior hybridization properties and water solubility. J Org Chem 76:5614–5627PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Brognara E, Fabbri E, Aimi F et al (2012) Peptide nucleic acids targeting miR-221 modulate p27Kip1 expression in breast cancer MDA-MB-231 cells. Int J Oncol 41:2119–2127PubMedGoogle Scholar
  12. 12.
    Kawano M, Kawazu C, Lizio M et al (2010) Reduction of non-insert sequence reads by dimer eliminator LNA oligonucleotide for small RNA deep sequencing. Biotechniques 49:751–755PubMedCrossRefGoogle Scholar
  13. 13.
    Blondal T, Jensby NS, Baker A et al (2013) Assessing sample and miRNA profile quality in serum and plasma or other biofluids. Methods 59:S1–S6PubMedCrossRefGoogle Scholar
  14. 14.
    Orom UA, Kauppinen S, Lund AH (2006) LNA-modified oligonucleotides mediate specific inhibition of microRNA function. Gene 372:137–141PubMedCrossRefGoogle Scholar
  15. 15.
    Wang D, Zhang Z, O’Loughlin E et al (2013) MicroRNA-205 controls neonatal expansion of skin stem cells by modulating the PI(3)K pathway. Nat Cell Biol 15:1153–1163PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Singh U, Keirstead N, Wolujczyk A et al (2013) General principles and methods for routine automated microRNA in situ hybridization and double labeling with immunohistochemistry. Biotech Histochem 89(4):259–266PubMedCrossRefGoogle Scholar
  17. 17.
    Jorgensen S, Baker A, Moller 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
  18. 18.
    Nielsen BS (2012) MicroRNA in situ hybridization. In: Fan JB (ed) Next-generation microRNA expression profiling technology: methods and protocols, vol 822, Methods in molecular biology. Humana Press, New York, pp 67–84CrossRefGoogle Scholar
  19. 19.
    Nielsen BS, Holmstrom K (2013) Combined microRNA in situ hybridization and immunohistochemical detection of protein markers. In: Moll F, Colombo R (eds) Target identification and validation in drug discovery, vol 986, Methods in molecular biology. Humana Press, New York, pp 353–365CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Boye Schnack Nielsen
    • 1
    Email author
  • Trine Møller
    • 1
  • Kim Holmstrøm
    • 1
  1. 1.Molecular HistologyBioneer A/SHørsholmDenmark

Personalised recommendations