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In situ localization of small RNAs in plants by using LNA probes


Small RNAs have crucial roles in numerous aspects of plant biology. Despite our current understanding of their biogenesis and mechanisms of action, the biological function of small RNAs, particularly miRNAs, remains largely unknown. To decipher small RNA function, knowledge about their spatiotemporal patterns of expression is essential. Here we report an in situ hybridization method for the precise localization of small RNAs in plants by using locked nucleic acid (LNA) oligonucleotide probes. This method has been adapted from protocols used to detect messenger RNAs in formaldehyde-fixed and paraffin-embedded tissue sections, but it includes essential optimizations in key prehybridization, hybridization and posthybridization steps. Most importantly, optimization of probe concentration and hybridization temperature is required for each unique LNA probe. We present the detailed protocol starting from sectioned tissues, and we include troubleshooting tips and recommended controls. This method has been used successfully in several plant species and can be completed within 2–6 d.

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Figure 1: Optimization of LNA probe concentration and hybridization temperature is essential for the successful in situ localization of small RNAs.
Figure 2: Schematic overview of the major steps in the protocol for the in situ detection of small RNAs using LNA oligo probes.

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We thank F. Nogueira and D. Chitwood for their initial contributions to the development of this protocol; M. Scanlon and K. Petsch for helpful suggestions and A. Husbands for the critical review of the manuscript. Research in the laboratory of M.C.P.T. is supported by grants DBI-0820610 and IOS-1022102 from the US National Science Foundation.

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M.J. developed the protocol and performed all experiments. M.J. and M.C.P.T. contributed to the design and analysis of experiments and wrote the paper.

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Correspondence to Marja C P Timmermans.

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The authors declare no competing financial interests.

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Javelle, M., Timmermans, M. In situ localization of small RNAs in plants by using LNA probes. Nat Protoc 7, 533–541 (2012).

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