Single Molecule RNA FISH in Root Cells.

Methods that allow the study of gene expression regulation are continually advancing. Here, we present an hybridization protocol capable of detecting individual mRNA molecules in plant root cells, thus permitting the accurate quantification and localization of mRNA within fixed samples ( Duncan , 2016 ; Rosa , 2016 ). This single molecule RNA fluorescence hybridization (smFISH) uses multiple single-labelled oligonucleotide probes to bind target RNAs and generate diffraction-limited signals that can be detected using a wide-field fluorescence microscope. We adapted a recent version of this method that uses 48 fluorescently labeled DNA oligonucleotides (20 mers) to hybridize to different portions of each transcript ( Raj , 2008 ). This approach is simple to implement and has the advantage that it can be readily applied to any genetic background.