Max Planck Institute of Plant Physiology, Potsdam, Germany.
The James Hutton Institute, Dundee, UK.
Methods Mol Biol. 2023;2686:331-350. doi: 10.1007/978-1-0716-3299-4_17.
RNA in situ hybridization offers a means to study the spatial expression of candidate genes by making use of specific, labelled RNA probes on thin tissue sections. Unlike other methods, such as promoter GUS fusions, for which all regulatory sequences should be available and transgenic plants have to be generated, RNA in situ hybridization allows specific and direct detection of even low abundant transcripts at cellular resolution. Although various protocols exist, the results published throughout the literature indicate a very obvious problem of the technique: each step has the potential to affect the outcome, that is, the signal strength, presence or absence of background, and visibility of individual cells. The protocol described here tries to avoid all these problems by addressing each step in detail and providing advice regarding critical steps for a distinct visualization of gene expression on intact tissue sections without any background.
原位杂交技术提供了一种通过使用特定的、标记的 RNA 探针在薄组织切片上研究候选基因空间表达的方法。与其他方法（如启动子 GUS 融合）不同，后者需要所有的调控序列都可用，并生成转基因植物，原位杂交技术允许在细胞分辨率下特异性和直接检测即使是低丰度的转录本。尽管存在各种方案，但文献中发表的结果表明该技术存在一个非常明显的问题：每个步骤都有可能影响结果，即信号强度、背景的存在或不存在以及单个细胞的可见度。这里描述的方案试图通过详细解决每个步骤并提供关于在不产生任何背景的情况下在完整组织切片上明确可视化基因表达的关键步骤的建议来避免所有这些问题。
