Detection of MicroRNAs in Skin by In Situ Hybridization

MicroRNAs (miRNAs) are a family of small noncoding RNAs (~19-24 nt), expressed in a wide range of animals and plants. It is estimated that more than one-third of protein-encoding mRNAs are regulated by miRNAs. MiRNAs largely contribute to the regulation of gene expression by fine-tuning and buffering the activity of signaling pathways. Therefore, miRNAs represent remarkably diverse regulatory networks, playing a key role in the execution of gene expression programs in various cells and tissues. Many technical challenges have been encountered when investigating miRNAs, in particular, determining the spatiotemporal expression pattern of miRNAs in cells and tissues. Therefore, we describe here a well-established in situ hybridization (ISH) protocols for use of the detection and analysis of spatiotemporal expression patterns of miRNAs in skin and its appendages such as the hair follicle in both frozen and paraffin-embedded tissue sections. We describe in detail the different steps that are associated with utilizing ISH procedure on either frozen or paraffin-embedded tissues for miRNAs localization. Post-fixation of tissues, tissues are hybridized with LNA double-labeled probes with digoxygenin. Detection of hybridized probes is performed by using an alkaline phosphatase-coupled antibody against digoxygenin. The final step involves the use of substrates to develop the color of alkaline phosphatase-LNA-probe structure, leading to the identification of the spatiotemporal of target miRNAs in target tissue and cells. We also discuss two options for substrate color development in these procedures: (i) NBT/BCIP and (ii) BM-Purple. This method is a simple and convenient way of determining the spatiotemporal expression pattern of miRNAs, which has been a challenge since their discovery due to their relatively small size. Knowledge gained from ISH will be crucial for better understanding of individual miRNA(s) during distinct stages of development in various cells and tissues, and these protocols will be beneficial to the wider scientific community.