Highly Sensitive and Multiplexed Imaging of Short RNA Sequences in Formalin‐Fixed Paraffin‐Embedded Tissue

Sensitive and multiplexed imaging of short RNA sequences, including individual exons, exon junctions, small nuclear RNAs, circular RNAs, and other noncoding RNAs, is essential for understanding gene regulation and cellular heterogeneity in health and disease. However, such detection in formalin‐fixed paraffin‐embedded (FFPE) tissue remains difficult due to RNA fragmentation, low abundance, and strong tissue autofluorescence. Here, we present a specific amplification‐triggered amplification fluorescence in situ hybridization (SATA‐FISH) platform optimized for multiplexed imaging of short RNA targets in FFPE tissue. The method integrates branched DNA amplification with enzymatic tyramide signal amplification to achieve exceptional sensitivity, while dual‐probe recognition and checkpointed amplification ensure high specificity. Cleavable fluorescent tyramide chemistry enables iterative staining and signal removal, allowing repeated rounds of multiplexed imaging of the same sample. Using as few as three probe pairs, the system reliably detects RNA sequences <150 nucleotides with high signal‐to‐noise ratios. We validated this approach by imaging distinct regions of Ubiquitin C transcripts in FFPE human brain tissue. Comparative analysis with RNAscope on the same tissue demonstrated strong consistency in transcript quantification. SATA‐FISH provides a robust platform for sensitive, specific, and multiplexed imaging of short RNA sequences in clinically relevant FFPE tissue.