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

Nature Protocols
Marie Javelle, Marja C P Timmermans

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.

References

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Citations

Mar 6, 2004·Nature·Michelle T JuarezMarja C P Timmermans
Sep 6, 2005·Science·Cheng LuPamela J Green
Dec 22, 2005·Nature Methods·Wigard P KloostermanRonald H A Plasterk
May 4, 2006·Annual Review of Plant Biology·Matthew W Jones-RhoadesBonnie Bartel
Apr 4, 2007·Genes & Development·Fabio T S NogueiraMarja C P Timmermans
Apr 5, 2007·Nature Protocols·Philip B BrewerEva Benková
Jun 19, 2007·Trends in Plant Science·Ramanjulu SunkarJian-Kang Zhu
Feb 16, 2008·Nature Protocols·Patricia A MaroneyTimothy W Nilsen
Apr 5, 2008·Nature Protocols·Chang-Gong LiuCarlo M Croce
Jan 3, 2009·PLoS Genetics·Fabio T S NogueiraMarja C P Timmermans
Mar 10, 2009·Genes & Development·Daniel H ChitwoodMarja C P Timmermans
Aug 25, 2009·Current Opinion in Plant Biology·Ignacio Rubio-SomozaJames C Carrington
Apr 23, 2010·Nature·Annelie CarlsbeckerPhilip N Benfey
Mar 25, 2011·Methods in Molecular Biology·Ericka R Havecker
Apr 21, 2011·Journal of Experimental Botany·Chui E WongPrem L Bhalla

Related Concepts

Establishment and Maintenance of Localization
Nucleic Acid Hybridization Procedure
Oligonucleotide Probes
locked nucleic acid
Oligonucleotides
alpha-L-LNA
Anabolism
RNA, Small Untranslated
MicroRNAs
Paraffin

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