Whole mount RNA-FISH on ovules and developing seeds

Abstract

A key element to understand developmental and reproductive processes like germline development, double fertilization, and embryogenesis is the study of cell-specific gene expression patterns which is best analyzed by RNA in situ hybridization. Different visualization techniques have been established to mark either the region of mRNA production (using the classical chromogenic detection system) or the specific localization of mRNAs (using fluorescent labeled probes). In this chapter, we describe and compare whole mount RNA in situ hybridization techniques on ovules and young developing seeds from Arabidopsis thaliana using three different detection systems. The alkaline phosphatase (AP) coupled antibody detecting the antigen labeled probe facilitates the production of a precipitating dye indicating mRNA presence: (1) using BCIP/NBT as substrates, it is converted to a blue staining that can be visualized using differential interference contrast (DIC) microscopy. Alternatively, (2) using Fast-Red as a substrate it is converted to a purple fluorescent staining that can be visualized either by light microscopy or, for a higher cellular resolution, by confocal microscope. To analyze mRNA distribution with subcellular resolution we (3) describe a third, highly sensitive fluorescent detection system, which is based on the enzymatic activity of a peroxidase. In combination with a tyramide signal amplification (TSA) system, it leads to multi-fluorescent labeled antibodies marking the mRNA bound probe locally.