Detection of microRNAs in Plants by In Situ Hybridisation

MicroRNAs (miRNAs) are short, about 21 nucleotides in length, non-coding, regulatory RNA molecules representing a new layer in post-transcriptional gene expression regulation. Spatial and temporal analysis of miRNA accumulation by in situ analyses is the prerequisite of understanding the precise biological functions of miRNAs. Since miRNAs are very short molecules, their in situ analysis is technically demanding. Our method is based on the usage of highly sensitive LNA-modified oligonucleotide probes. LNA modification significantly enhances the sensitivity and specificity of miRNA detecting probes and provides relatively easy in situ miRNA detection. Here, we describe a protocol for this challenging technique step by step, in order to help every user to achieve success.