Francoz Edith, Ranocha Philippe, Dunand Christophe, Burlat Vincent
Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, Castanet Tolosan, France.
Methods Mol Biol. 2019;1933:99-130. doi: 10.1007/978-1-4939-9045-0_6.
(m)RNA spatiotemporal pattern of distribution is of key importance to decipher gene function. In this post-genomic era, numerous transcriptomic studies are made publicly available, sometimes reaching a tissular resolution and even more rarely the cellular level. This "one tissue-numerous genes" information can be completed by the reverse "one gene-numerous tissues" picture through traditional RNA in situ hybridization (ISH). Here, we present a method including (1) principles of transcriptomic data mining to be performed prior and following ISH and (2) a detailed step-by-step medium-throughput ISH protocol performed on serial sections from tissue microarrays. In a recent work, we implemented this method for 39 selected genes studied by medium-throughput ISH complementing an existing tissue-specific transcriptomic dataset focused on the model plant Arabidopsis seed development kinetics (Francoz et al., Scientific Reports 6:24644, 2016). This full integration of ISH and transcriptomics demonstrated the complementarity of both techniques in terms of tissue/cell specificity, signal sensitivity, gene specificity, and spatiotemporal resolution.
（信使）核糖核酸的时空分布模式对于解读基因功能至关重要。在这个后基因组时代，大量转录组学研究成果已公开，有时能达到组织分辨率，而达到细胞水平的则更为罕见。这种“一个组织-众多基因”的信息可以通过传统的RNA原位杂交（ISH），以相反的“一个基因-众多组织”图景来补充。在此，我们展示一种方法，包括（1）在ISH之前和之后进行的转录组数据挖掘原则，以及（2）在组织微阵列的连续切片上进行的详细的中通量ISH分步方案。在最近的一项工作中，我们将此方法应用于通过中通量ISH研究的39个选定基因，补充了一个现有的专注于模式植物拟南芥种子发育动力学的组织特异性转录组数据集（弗朗索瓦等人，《科学报告》6:24644, 2016）。ISH和转录组学的这种完全整合在组织/细胞特异性、信号敏感性、基因特异性和时空分辨率方面证明了这两种技术的互补性。
