title: Fluorescent whole-mount RNA in situ hybridization (F-WISH) in plant germ cells and the fertilized ovule.
abstract: First evidence on gene function and regulation is provided by the cellular expression pattern in complex tissues. However, to understand the activity of a specific gene, it is essential to analyze the regulatory network, which controls the spatio-temporal translation pattern during the entire life span of the transcribed mRNA. To explore mechanisms which control mRNA abundance and localization in space and time, it is necessary to visualize mRNAs quantitatively with a subcellular resolution, without sectioning the tissues. We have adapted and optimized a protocol for colorimetric whole-mount RNA in situ hybridization (WISH) using egg cell-specific digoxigenin (DIG) labeled probes (Hejátko et al., 2006) [1] on ovules and early seeds of Arabidopsis. Furthermore, we established a fluorescent whole-mount RNA in situ hybridization (F-WISH) protocol, which allows mRNA visualization on a subcellular level. The polar localized mRNA of SBT4.13, encoding a subtilase, was identified using this protocol. Both methods are described and discussed in detail. Additionally a (F)-WISH flow-chart is provided along with a troubleshooting table.
authors.affiliation: Cell Biology and Plant Biochemistry, Biochemie-Zentrum Regensburg, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany. Electronic address: andrea.bleckmann@ur.de.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bleckmann
authors.affiliation: Cell Biology and Plant Biochemistry, Biochemie-Zentrum Regensburg, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany. Electronic address: thomas.dresselhaus@ur.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Dresselhaus
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure*; D004076:Digoxigenin / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D055993:Germ Cells, Plant / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D056771:Ovule / Q000378:metabolism / Q000648:ultrastructure*; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D059629:Signal-To-Noise Ratio; D013381:Subtilisins / Q000737:chemistry; D016707:Tissue Fixation / Q000379:methods; D014158:Transcription, Genetic; D014439:Tyramine / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D012333:RNA, Messenger; D013381:Subtilisins; C119297:subtilase, plant; D004076:Digoxigenin; D014439:Tyramine
keywords: F-WISH; Ovule; Subtilase; TSA System; WISH; mRNA