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