Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
Nat Protoc. 2012 Feb 23;7(3):533-41. doi: 10.1038/nprot.2012.006.
Small RNAs 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 RNAs, particularly miRNAs, remains largely unknown. To decipher small RNA function, knowledge about their spatiotemporal patterns of expression is essential. Here we report an in situ hybridization method for the precise localization of small RNAs in plants by using locked nucleic acid (LNA) oligonucleotide probes. This method has been adapted from protocols used to detect messenger RNAs 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 LNA 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.
小 RNA 在植物生物学的众多方面发挥着关键作用。尽管我们目前已经了解了它们的生物发生和作用机制，但小 RNA，特别是 miRNA 的生物学功能在很大程度上仍然未知。为了阐明小 RNA 的功能，了解它们时空表达模式是必不可少的。在这里，我们报告了一种使用锁核酸 (LNA) 寡核苷酸探针在植物中精确定位小 RNA 的原位杂交方法。该方法是从用于检测福尔马林固定和石蜡包埋组织切片中信使 RNA 的方案改编而来，但在关键的预杂交、杂交和杂交后步骤中进行了必要的优化。最重要的是，每个独特的 LNA 探针都需要优化探针浓度和杂交温度。我们从切片组织开始介绍详细的方案，并包括故障排除技巧和推荐的对照。该方法已在几种植物物种中成功使用，可在 2-6 天内完成。
