Analysis of 3D gene expression patterns in plants using whole-mount RNA in situ hybridization.

IF: 17.021

Cited by: 27

Abstract

In situ mRNA hybridization is one of the most powerful techniques for analyzing patterns of gene expression. However, its usefulness is limited in complex plant tissues by the need to fix, embed and section samples before localizing the desired mRNA. Here we present a robust whole-mount in situ hybridization method that allows easy access to patterns of gene expression in intact, complex tissues, such as the inflorescence apex of Arabidopsis thaliana. The tissue is first fixed and then permeabilized by treatment with a cocktail of cell wall-digesting enzymes that has been optimized to preserve the integrity of tissue structures, while also permitting the detection of expression patterns in deep tissues. In addition to colorimetric staining, fluorimetric staining that can be imaged by confocal microscopy can also be used with this protocol, thus providing full 3D resolution. The entire procedure can take <3 d from tissue preparation to image acquisition.

Keywords

Gene Expression

PROCEDURE

MeSH terms

Arabidopsis

Colorimetry

Gene Expression Profiling

Imaging, Three-Dimensional

In Situ Hybridization

Microscopy, Confocal

Plants

Polymerase Chain Reaction

RNA, Plant

Authors

Rozier, Frédérique

Mirabet, Vincent

Vernoux, Teva

Das, Pradeep

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