Abstract
Here we describe a method for sensitive and specific histological detection of microRNAs (miRNAs) by in situ hybridization. The protocol focuses on the use of locked nucleic acids (LNAs), which are bi-cyclic RNA analogs that allow a significant increase in the hybridization temperature and thereby an enhanced stringency for short probes as required for miRNA detection. The protocol is optimized for cryosections in order to study the spatial and temporal expression of miRNAs with high sensitivity and resolution. We detail how to construct probes, set up and conduct an LNA in situ hybridization experiment. In addition, we discuss alternative colorimetric strategies that can be used to effectively detect and visualize miRNAs including double staining with other markers. Setting up and conducting the in situ experiment is estimated to take ∼1 week, assuming that all the component parts are readily available.
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References
Schaeren-Wiemers, N. & Gerfin-Moser, A. A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes. Histochemistry 100, 431–440 (1993).
Jain, K.K. Current status of fluorescent in situ hybridisation. Med. Device Technol. 15, 14–17 (2004).
Hargrave, M., Bowles, J. & Koopman, P. In situ hybridization of whole-mount embryos. Methods Mol. Biol. 326, 103–113 (2006).
Kidner, C. & Timmermans, M. In situ hybridization as a tool to study the role of microRNAs in plant development. Methods Mol. Biol. 342, 159–179 (2006).
Berezikov, E. & Plasterk, R.H. Camels and zebrafish, viruses and cancer: a microRNA update. Hum. Mol. Genet. 14 Spec No. 2, R183–R190 (2005).
Engels, B.M. & Hutvagner, G. Principles and effects of microRNA-mediated post-transcriptional gene regulation. Oncogene 25, 6163–6169 (2006).
Du, T. & Zamore, P.D. microPrimer: the biogenesis and function of microRNA. Development 132, 4645–4652 (2005).
Kim, V.N. MicroRNA biogenesis: coordinated cropping and dicing. Nat. Rev. Mol. Cell Biol. 6, 376–385 (2005).
Ambros, V., Lee, R.C., Lavanway, A., Williams, P.T. & Jewell, D. MicroRNAs and other tiny endogenous RNAs in C. elegans . Curr. Biol. 13, 807–818 (2003).
Lagos-Quintana, M. et al. Identification of tissue-specific microRNAs from mouse. Curr. Biol. 12, 735–739 (2002).
Miska, E.A. et al. Microarray analysis of microRNA expression in the developing mammalian brain. Genome Biol. 5, R68 (2004).
Kloosterman, W.P., Wienholds, E., de Bruijn, E., Kauppinen, S. & Plasterk, R.H. In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat. Methods 3, 27–29 (2006).
Wienholds, E. et al. MicroRNA expression in zebrafish embryonic development. Science 309, 310–311 (2005).
Valoczi, A. et al. Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res. 32, e175 (2004).
Obernosterer, G., Leuschner, P.J., Alenius, M. & Martinez, J. Post-transcriptional regulation of microRNA expression. RNA 12, 1161–1167 (2006).
Petersen, M. et al. The conformations of locked nucleic acids (LNA). J. Mol. Recogn. 13, 44–53 (2000).
Bondensgaard, K. et al. Structural studies of LNA:RNA duplexes by NMR: conformations and implications for RNase H activity. Chemistry 6, 2687–2695 (2000).
Petersen, M., Nielsen, J.T., Bondensgaard, K., Wengel, J. & Jacobsen, J.P. Structural characterization of LNA and alpha-L-LNA hybridized to RNA. Nucleosides Nucleotides Nucleic Acids 22, 1691–1693 (2003).
Thomsen, R., Nielsen, P.S. & Jensen, T.H. Dramatically improved RNA in situ hybridization signals using LNA-modified probes. RNA 11, 1745–1748 (2005).
Hofacker, I.L. Vienna RNA secondary structure server. Nucleic Acids Res. 31, 3429–3431 (2003).
Rasmussen, K.R., Stoye, J. & Myers, E.W. Efficient q-gram filters for finding all epsilon-matches over a given length. J. Comput. Biol. 13, 296–308 (2006).
Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).
Acknowledgements
We thank Exiqon A/S, Denmark and especially Dr. Peter Roberts for the customized synthesis of miRCURY probes in order to study how sensitivity and specificity of miRNA detection using in situ hybridizations is affected depending on different LNA incorporations. Professor Renée Schroeder critically read the manuscript. J.M. is a Junior Group Leader at IMBA. M.A. was supported by a Marie Curie fellowship.
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Obernosterer, G., Martinez, J. & Alenius, M. Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections. Nat Protoc 2, 1508–1514 (2007). https://doi.org/10.1038/nprot.2007.153
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DOI: https://doi.org/10.1038/nprot.2007.153
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