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

Small RNARNARNAs 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 RNARNARNAs, particularly miRNARNARNAs, remains largely unknown. To decipher small RNARNARNA function, knowledge about their spatiotemporal patterns of expression is essential. Here we report an in situ hybridization method for the precise localization of small RNARNARNAs in plants by using locked nucleic acid (LNALNALNA)oligonucleotide probes. This method has been adapted from protocols used to detect messenger RNARNARNAs 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 LNALNALNA 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.