RNAscope

RNAscope is a proprietary RNA in situ hybridization method for detecting RNA transcripts in fixed tissues and cells with spatial resolution. Developed by Advanced Cell Diagnostics (ACD), now part of Akoya Biosciences, the technique provides high sensitivity and specificity by using a unique probe design and a cascade of amplification steps to visualize RNA molecules as discrete puncta.

Key to RNAscope is the double-Z probe design: each target region is interrogated by a pair of Z probes that must hybridize in tandem to form a binding site for a preamplifier, then amplifiers, and finally label probes. This "double-Z" requirement reduces off-target signal and yields strong, low-background staining. After tissue pretreatment to expose RNA, the probes are hybridized, and signals are developed using chromogenic or fluorescent detection.

RNAscope supports different sample types and multiplexing: it can be applied to formalin-fixed paraffin-embedded (FFPE) tissue, fresh-frozen tissue, or cultured cells, and can be combined with immunohistochemistry for protein detection. Multiplex RNAscope assays allow simultaneous detection of multiple RNA targets in the same sample, enabling cellular phenotyping and spatial mapping of gene expression.

Because results are visual counts of RNA puncta per cell, RNAscope provides semi-quantitative data and is dependent on sample quality and assay optimization. It is widely used in biomedical research and pathology for biomarker discovery, tumor profiling, and neuroscience studies, among other applications.