Appl Plant Sci. 2014 Apr 03;2(4). doi: 10.3732/apps.1400011. eCollection 2014 Apr.

Application of a novel and automated branched DNA in situ hybridization method for the rapid and sensitive localization of mRNA molecules in plant tissues.

Applications in plant sciences

Andrew J Bowling, Heather E Pence, Jeffrey B Church

PMID: 25202621 PMCID: PMC4103140 DOI: 10.3732/apps.1400011

Abstract

PREMISE OF THE STUDY: A novel branched DNA detection technology, RNAscope in situ hybridization (ISH), originally developed for use on human clinical and animal tissues, was adapted for use in plant tissue in an attempt to overcome some of the limitations associated with traditional ISH assays. •

METHODS AND RESULTS: Zea mays leaf tissue was formaldehyde fixed and paraffin embedded (FFPE) and then probed with the RNAscope ISH assay for two endogenous genes, phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PEPCK). Results from both manual and automated methods showed tissue- and cell-specific mRNA localization patterns expected from these well-studied genes. •

CONCLUSIONS: RNAscope ISH is a sensitive method that generates high-quality, easily interpretable results from FFPE plant tissues. Automation of the RNAscope method on the Ventana Discovery Ultra platform allows significant advantages for repeatability, reduction in variability, and flexibility of workflow processes.

Keywords: RNAscope; Zea mays; branched DNA; in situ hybridization; phosphoenolpyruvate carboxykinase; phosphoenolpyruvate carboxylase

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Publication Types

• Journal Article