title: Multi-probe in situ hybridization to whole mount Arabidopsis seedlings.
abstract: 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.
authors.affiliation: University of Calabria, Department of Ecology, Laboratory of Citofisiologia Vegetale, Arcavacata di Rende, Cosenza, Italy.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Bruno
authors.forename: Antonella
authors.initials: A
authors.lastname: Muto
authors.forename: Natasha D
authors.initials: ND
authors.lastname: Spadafora
authors.forename: Domenico
authors.initials: D
authors.lastname: Iaria
authors.forename: Adriana
authors.initials: A
authors.lastname: Chiappetta
authors.forename: Mieke
authors.initials: M
authors.lastname: Van Lijsebettens
authors.forename: Maria B
authors.initials: MB
authors.lastname: Bitonti
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics; D001710:Biotin; D004076:Digoxigenin; D019793:Fluorescein; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D051548:Histone Acetyltransferases / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics; D017403:In Situ Hybridization / Q000379:methods*; D026901:Membrane Transport Proteins / Q000235:genetics; D018613:Microscopy, Confocal / Q000379:methods; D009693:Nucleic Acid Hybridization / Q000379:methods; D015347:RNA Probes / Q000138:chemical synthesis* / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis*; D036226:Seedlings / Q000235:genetics*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D018398:Homeodomain Proteins; C092212:KNAT1 protein, Arabidopsis; D026901:Membrane Transport Proteins; C116363:PIN1 protein, Arabidopsis; D015347:RNA Probes; D012333:RNA, Messenger; D001710:Biotin; C502551:ELO3 protein, Arabidopsis; D051548:Histone Acetyltransferases; C521255:HUB1 protein, Arabidopsis; D044767:Ubiquitin-Protein Ligases; D004076:Digoxigenin; D019793:Fluorescein