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miRNA in situ hybridization in formaldehyde and EDC–fixed tissues

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Abstract

MicroRNAs are small regulatory RNAs with many biological functions and disease associations. We showed that in situ hybridization (ISH) using conventional formaldehyde fixation results in substantial microRNA loss from mouse tissue sections, which can be prevented by fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide that irreversibly immobilizes the microRNA at its 5′ phosphate. We determined optimal hybridization parameters for 130 locked nucleic acid probes by recording nucleic acid melting temperature during ISH.

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Figure 1: Visualization of miRNAs expressed at different levels in the mouse brain.
Figure 2: Formaldehyde and EDC–fixed sections show miRNA localized in the dendrites of neurons.

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Acknowledgements

We thank members of the Memorial Sloan Kettering Sequencing Core for 454 sequencing; S. Juranek, N. Renwick, J. McManus, H. Yamahachi and T. Farazi for comments on the manuscript; L. Fleming for the technical assistance; and C.D. Gilbert for his guidance. T.T. was supported by an Irma T. Hirschl Career Scientist Award, J.T.G.P. supported by a Kirschstein–National Research Service Award fellowship and the project was partly funded by US National Institutes of Health grants GM073047, EY18082-01A2 and MH080442.

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Author notes

  1. Janos Ludwig

    Present address: Present address: Institute of Clinical Chemistry and Pharmacology, University of Bonn, Sigmund Freud Str. 25, 53127 Bonn, Germany.,

Authors and Affiliations

  1. Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, Box 186, New York, 10065, New York, USA

    John T G Pena, Cherin Sohn-Lee, Sara H Rouhanifard, Janos Ludwig, Markus Hafner, Aleksandra Mihailovic, Cindy Lim, Daniel Holoch & Thomas Tuschl

  2. Weill Medical College of Cornell University, 1300 York Avenue, New York, 10021, New York, USA

    John T G Pena

  3. Biozentrum, University of Basel and Swiss Institute of Bioinformatics, Klingelbergstrasse 50-70, Basel, 4056, Switzerland

    Philipp Berninger & Mihaela Zavolan

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  1. John T G Pena
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Contributions

J.T.G.P., S.H.R. and C.S.-L. designed LNA-modified probes, conducted the mouse ISH experiments and recorded ISH images using microscopy. C.S.-L. recorded the melting profiles for the miRNA-LNA probe duplexes. C.S.-L., C.L., D.H. and A.M. synthesized LNA probes. S.H.R. sectioned all tissues. J.T.G.P. dissected the mouse brain sections for ISH, and M.H. and A.M. prepared RNA for large-scale small RNA sequencing. J.L. advised on chemical conditions used for the miRNA cross-linking. M.Z. and P.B. designed and carried out the small RNA annotation. J.T.G.P. and T.T. wrote the manuscript.

Corresponding author

Correspondence to Thomas Tuschl.

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Competing interests

T.T. is a co-founder and scientific advisor of Alnylam Pharmaceuticals and a scientific advisor of Regulus Therapeutics.

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Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1–3, Supplementary Methods (PDF 11132 kb)

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Pena, J., Sohn-Lee, C., Rouhanifard, S. et al. miRNA in situ hybridization in formaldehyde and EDC–fixed tissues. Nat Methods 6, 139–141 (2009). https://doi.org/10.1038/nmeth.1294

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  • DOI: https://doi.org/10.1038/nmeth.1294

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