Duplex In Situ Hybridization of Virus Nucleic Acids in Plant Tissues Using RNAscope

RNAscope has been recently introduced by Advanced Cell Diagnostics (Newark, CA, USA) for in situ hybridization (ISH) of target RNAs using a proprietary technology for probe design and hybridization assay. The method has been extensively used as a basis for sensitive diagnostic assays in the medical field, while applications of this technique in plant sciences are still rare. Here, we describe a multiplex ISH protocol for detection of two plant viruses in formalin-fixed paraffin-embedded tissue sections from cassava. The dual-color protocol described can be used as reference for virus/host interaction studies including the visualization of virus nucleic acids and plant endogenous mRNAs. RNAscope provides a specificity and sensitivity of target detection that otherwise cannot be reached.