RNA In Situ Hybridization on Plant Tissue Sections: Expression Analysis at Cellular Resolution

RNA in situ hybridization offers a means to study the spatial expression of candidate genes by making use of specific, labelled RNA probes on thin tissue sections. Unlike other methods, such as promoter GUS fusions, for which all regulatory sequences should be available and transgenic plants have to be generated, RNA in situ hybridization allows specific and direct detection of even low abundant transcripts at cellular resolution. Although various protocols exist, the results published throughout the literature indicate a very obvious problem of the technique: each step has the potential to affect the outcome, that is, the signal strength, presence or absence of background, and visibility of individual cells. The protocol described here tries to avoid all these problems by addressing each step in detail and providing advice regarding critical steps for a distinct visualization of gene expression on intact tissue sections without any background.