The RNAscope principle. Target probe hybridization: Sets of oligonucleotide probes specific for different mRNAs are designed, such that each pair of two z-shaped probes (each approximately 20 bp) hybridizes to the specific target mRNA. In total, 20 pairs are designed for each RNA of interest. In addition to the complementary RNA sequence, each probe contains a spacer, as well as a tail sequence to which the pre-amplifier structure binds in the next step. Signal amplification: As a result of pre-amplifier interaction with the tail sequence, a tree-like scaffold that provides multiple binding sites for fluorescent labels is formed. Fluorescent labeling: Subsequently, distinct fluorescent labels are simultaneously added. Together, the large number of fluorescent labels bound to each target probe pair provides a strong signal, allowing detection of rare mRNA species.