In situ localization of small RNAs in plants by using LNA probes
In situ localization of small RNAs in plants by using LNA probes
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DOI：
10.1038/nprot.2012.006
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发表时间：
2012-03-01
期刊：
影响因子：
14.8
通讯作者：
Timmermans, Marja C. P.
中科院分区：
文献类型：
--
作者：
Javelle, Marie;Timmermans, Marja C. P.
小RNA在植物生物学的许多方面起着至关重要的作用。尽管我们目前对它们的生物发生和作用机制有了了解，但小RNA，特别是miRNAs的生物学功能在很大程度上仍然不清楚。要破译小RNA的功能，了解它们的时空表达模式是必不可少的。在这里，我们报告了一种利用锁定的核酸(LNA)寡核苷酸探针对植物中的小RNA进行精确定位的原位杂交方法。这种方法是从用于检测甲醛固定和石蜡包埋组织切片中的信使RNA的方案改编而来的，但它包括关键的预杂交、杂交和后杂交步骤的基本优化。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。我们介绍了从切片组织开始的详细方案，并包括故障排除提示和推荐的控制措施。该方法已在几种植物上成功应用，可在2-6d内完成。
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.