Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kloosterman WP, Wienholds E, de Bruijn E (2006) In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat Methods 3:27–29
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 2:2520–2528
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
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 4:107–115
Pena JTG, Sohn-Lee C, Rouhanifard SH et al (2009) miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat Methods 6:139–141
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 52:375–381
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 16:4246–4255
Nielsen BS (2012) MicroRNA in situ hybridization. Methods Mol Biol 822:67–84
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/JCI68760
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 54:3607–3630
Vester B, Wengel J (2004) LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43:13233–13241
McTigue PM, Peterson RJ, Kahn JD (2004) Sequence-dependent thermodynamic parameters for locked nucleic acid (LNA)-DNA duplex formation. Biochemistry 43:5388–5405
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 59:661–672
Nielsen BS, Holmstrøm K (2013) Combined microRNA in situ hybridization and immunohistochemical detection of protein markers. Methods Mol Biol 986:353–365
Acknowledgments
The authors thank the Danish Ministry and Agency of Science, Technology, and Innovation for funding to MJS.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Søe, M.J., Dufva, M., Holmstrøm, K. (2014). Detection of Small Noncoding RNAs by In Situ Hybridization Using Probes of 2′-O-Methyl RNA + LNA. In: Werner, A. (eds) Animal Endo-SiRNAs. Methods in Molecular Biology, vol 1173. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0931-5_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0931-5_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0930-8
Online ISBN: 978-1-4939-0931-5
eBook Packages: Springer Protocols