Localization of miRNAs by In Situ Hybridization in Plants Using Conventional Oligonucleotide Probes

Among the epigenetic mechanisms studied with a greater interest in the last decade are the microRNAs (miRNAs). These small noncoding RNA sequences that are approximately 17-22 nucleotides in length play an essential role in many biological processes of various organisms, including plants. The analysis of spatiotemporal expression of miRNAs provides a better understanding of the role of these small molecules in plant development, cell differentiation, and other processes; but such analysis is also an important method for the validation of biological functions. In this work, we describe the optimization of an efficient protocol for the spatiotemporal analysis of miRNA by in situ hybridization using different plant tissues embedded in paraffin. Instead of LNA-modified probes that are typically used for this work, we use conventional oligonucleotide probes that yield a high specificity and clean distribution of miRNAs.