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Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections

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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Figure 1: miR-138 in situ staining of mouse hippocampus.
Figure 2: LNA-dependent hybridization stringency of miR-138.
Figure 3: In situ hybridization experiment flowchart.
Figure 4: Detection of miRNA maturation stages by LNA probes.

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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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Correspondence to Mattias Alenius.

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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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