Multiple gene detection by in situ RT-PCR in isolated plant cells and tissues

With the number of functional genomic approaches in plant biology increasing daily, the demand for rapid and reliable RNA localization techniques for gene characterization is being felt. We present herein a novel, liquid phase in situ RT-PCR (IS-RT-PCR) protocol using a combination of gene-specific fluorescent primers and spectral confocal microscopy to localize target RNA in epicotyl sections and xylogenic suspension cultures of Zinnia elegans. Potential sources of artefacts from fixation to gene detection were systematically eliminated using both fluorescent primers and nucleotides for 18S rRNA gene detection, resulting in a set of optimal parameters for IS-RT-PCR that may be readily adapted to any target gene. By judiciously choosing fluorescent primers with non-overlapping fluorochromes, we have shown that our technique is readily adapted to multiplex IS-RT-PCR, enabling the simultaneous localization of more than one gene within a complex tissue or heterogeneous cell population. A 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein (6-HEX)-labelled primer and a tetrachloro-6-carboxy-fluorescein (TET)-labelled primer were designed for two marker genes associated with programmed cell death in tracheary elements (TEs): an endonuclease (Zen1) and a cysteine protease (ZcP4), respectively. An additional Cyan5 (Cy5)-labelled primer was used to monitor 18SrRNA expression. As expected, the 18S signal was constitutively expressed throughout epicotyls sections and living cells in xylogenic in vitro cultures, whereas Zen1 and ZcP4 were co-localized in forming TEs both in planta and in vitro. Analogous to clustering analysis of gene expression using microarrays to elucidate common metabolic pathways and developmental processes, this novel technique is perfectly adapted to gaining a better understanding of gene function via the coordinated expression of genes in specific cell types of complex tissues and cell populations.