DOW RESTRICTED Application of a Novel and Automated in situ Hybridization Method for the Rapid and Sensitive Localization of mRNA Molecules in Plant Tissues

Localization of specific mRNA molecules within large and diverse sample sets is often hampered by the limitations of traditional in situ hybridization (ISH) methods. RNAscope ISH is an improved method for localizing mRNA molecules more specifically, more rapidly, and with greater sensitivity than previously possible with conventional ISH methods. RNAscope ISH utilizes pairs of ‘Z-probes’ that are highly specific to target genes and yet are small enough to readily diffuse into tissue sections. Each Z-probe is comprised of a unique region of around 25 bases that is complementary to the mRNA of interest, a short linker region, and one half of a split ‘PreAmp’ binding site. The successful hybridization of both members of a Z-probe pair side-by-side on a target mRNA allows the binding of a PreAmp molecule (Fig. 1). Bound PreAmp molecules allow the binding of Amp molecules, which in turn allow binding of label molecules, which either catalyze the deposition of chromogen (e.g., DAB) or are directly detected by microscopy (e.g., fluorescein) [1]. The signal detected by microscopy is thus both highly specific and highly amplified.