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

Javelle, M., Timmermans, M. C. (February 2012) In situ localization of small RNAs in plants by using LNA probes. Nat Protoc, 7 (3). pp. 533-41. ISSN 1750-2799 (Electronic)1750-2799 (Linking)

URL: http://www.ncbi.nlm.nih.gov/pubmed/22362159
DOI: 10.1038/nprot.2012.006

Abstract

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.

Item Type: Paper
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing
bioinformatics > genomics and proteomics
organism description > plant
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > sRNA
CSHL Authors:
Communities: CSHL labs > Timmermans lab
CSHL Post Doctoral Fellows
Depositing User: Matt Covey
Date: 23 February 2012
Date Deposited: 30 Jan 2013 19:17
Last Modified: 02 May 2013 18:57
PMCID: PMC3308598
Related URLs:
URI: http://repository.cshl.edu/id/eprint/26971

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