Bruno Leonardo, Muto Antonella, Spadafora Natasha D, Iaria Domenico, Chiappetta Adriana, Van Lijsebettens Mieke, Bitonti Maria B
University of Calabria, Department of Ecology, Laboratory of Citofisiologia Vegetale, Arcavacata di Rende, Cosenza, Italy.
Int J Dev Biol. 2011;55(2):197-203. doi: 10.1387/ijdb.103132lb.
In situ RNA-RNA hybridization (ISH) is a molecular method for localization of gene transcripts at the cellular level and is widely used to provide spatial and temporal information regarding gene expression. However, standard protocols are complex and laborious to implement, restricting analysis to one or a few genes at any one time, each one observed on separate ISH preparations. Multi-probe whole-mount in situ hybridization is a powerful technique to compare the expression patterns of two or more genes simultaneously in the same tissue or organ. We describe for the first time in plants, the detection of three different mRNAs in a single fixed whole mount Arabidopsis seedling. A combination of bright fluorescent secondary antibodies was used for the detection of riboprobes differentially labeled by digoxigenin, biotin and fluorescein. The 3-D detection of each of the multiple fluorescent hybridization signals or in combination was obtained through confocal laser-scanning microscopy. The reliability of the method was tested in the root, using the PINFORMED (PIN) genes with non-overlapping temporal and spatial expression patterns. In the shoot, a class-I KNOTTED -like homeobox gene from Arabidopsis (KNAT1) with expression restricted to the shoot apical meristem was used in combination with ELONGATOR3 (ELO3) gene. In addition, the expression patterns of ELONGATOR complex gene (ELO2, ELO3) and HISTONE MONOUBIQUITINATION1 (HUB1) genes were analyzed in both shoot and root and a partial overlapping was observed. The whole procedure takes only 6 days.
原位RNA-RNA杂交（ISH）是一种在细胞水平定位基因转录本的分子方法，被广泛用于提供有关基因表达的时空信息。然而，标准方案实施起来复杂且费力，限制了在任何一个时间对一个或几个基因的分析，每个基因都在单独的ISH制剂上观察。多探针整装原位杂交是一种强大的技术，可同时比较同一组织或器官中两个或更多基因的表达模式。我们首次在植物中描述了在单个固定的拟南芥整株幼苗中检测三种不同的mRNA。使用明亮荧光二抗组合来检测用地高辛、生物素和荧光素差异标记的核糖探针。通过共聚焦激光扫描显微镜获得多个荧光杂交信号中每个信号的三维检测结果或其组合检测结果。该方法的可靠性在根中进行了测试，使用了具有不重叠时空表达模式的PINFORMED（PIN）基因。在地上部分，将拟南芥中一个表达局限于茎尖分生组织的I类KNOTTED样同源异型盒基因（KNAT1）与ELONGATOR3（ELO3）基因结合使用。此外，还分析了ELONGATOR复合基因（ELO2、ELO3）和组蛋白单泛素化1（HUB1）基因在地上部分和根中的表达模式，观察到部分重叠。整个过程仅需6天。
