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Methods. 2016 Apr 1;98:66-73. doi: 10.1016/j.ymeth.2015.10.019. Epub 2015 Oct 30.

Fluorescent whole-mount RNA in situ hybridization (F-WISH) in plant germ cells and the fertilized ovule.

Author information

1
Cell Biology and Plant Biochemistry, Biochemie-Zentrum Regensburg, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany. Electronic address: andrea.bleckmann@ur.de.
2
Cell Biology and Plant Biochemistry, Biochemie-Zentrum Regensburg, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany. Electronic address: thomas.dresselhaus@ur.de.

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.

KEYWORDS:

F-WISH; Ovule; Subtilase; TSA System; WISH; mRNA

PMID:
26521978
DOI:
10.1016/j.ymeth.2015.10.019
[Indexed for MEDLINE]

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