School of Science and Technology, Nottingham Trent University, Nottingham, UK.
Methods Mol Biol. 2020;2154:187-196. doi: 10.1007/978-1-0716-0648-3_16.
MicroRNAs (miRNAs) are a family of small noncoding RNAs (~19-24 nt) 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. We describe here a well-established in situ hybridization protocol for 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 in situ hybridization procedure on either frozen or paraffin-embedded tissues for miRNAs localization. Postfixation, 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 identification of the spatiotemporal location of target miRNAs in target tissue and cells. We also discuss two options for substrate color development in these procedures: (1) NBT/BCIP and (2) 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 in situ hybridization is crucial for better understanding of the roles of individual miRNA(s) during distinct stages of development in various cells and tissues. These protocols will be beneficial to the wider scientific community.
MicroRNAs (miRNAs) 是一类小型非编码 RNA（~19-24nt），在各种细胞和组织中基因表达程序的执行中发挥关键作用。在研究 miRNAs 时遇到了许多技术挑战，特别是确定 miRNAs 在细胞和组织中的时空表达模式。我们在这里描述了一种成熟的原位杂交技术，用于检测和分析皮肤及其附属物（如毛囊）中 miRNAs 的时空表达模式，包括冷冻和石蜡包埋组织切片。我们详细描述了与在冷冻或石蜡包埋组织上进行原位杂交程序相关的不同步骤，以用于 miRNAs 定位。固定后，将组织与带有 digoxigenin 的 LNA 双标记探针杂交。通过使用针对 digoxigenin 的碱性磷酸酶偶联抗体检测杂交探针。最后一步涉及使用底物来开发碱性磷酸酶-LNA-探针结构的颜色，从而鉴定目标组织和细胞中目标 miRNAs 的时空位置。我们还讨论了这两种程序中底物显色的两种选择：(1) NBT/BCIP 和 (2) BM Purple。由于它们相对较小的尺寸，该方法是确定 miRNAs 时空表达模式的一种简单方便的方法，这一直是自发现以来的一个挑战。通过原位杂交获得的知识对于更好地理解单个 miRNA 在各种细胞和组织的不同发育阶段中的作用至关重要。这些方案将对更广泛的科学界有益。
