Multi-probe in situ hybridization to whole mount Arabidopsis seedlings

In situ RNA-RNA hybridization (ISH) is a molecular method for localization of genetranscripts at the cellular level and is widely used to provide spatial and temporal informationregarding 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 separateISH preparations. Multi-probe whole-mount in situ hybridization is a powerful technique tocompare the expression patterns of two or more genes simultaneously in the same tissue ororgan. We describe for the first time in plants, the detection of three different mRNAs in a singlefixed whole mount Arabidopsis seedling. A combination of bright fluorescent secondary antibodieswas used for the detection of riboprobes differentially labeled by digoxigenin, biotin andfluorescein. The 3-D detection of each of the multiple fluorescent hybridization signals or incombination was obtained through confocal laser-scanning microscopy. The reliability of themethod was tested in the root, using the PINFORMED (PIN) genes with non-overlapping temporaland spatial expression patterns. In the shoot, a class-I KNOTTED -like homeobox gene fromArabidopsis (KNAT1) with expression restricted to the shoot apical meristem was used incombination with ELONGATOR3 (ELO3) gene. In addition, the expression patterns of ELONGATORcomplex gene (ELO2, ELO3) and HISTONE MONOUBIQUITINATION1 (HUB1) genes were analyzedin both shoot and root and a partial overlapping was observed. The whole procedure takes only6 days.