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.