Summary
Advances in precision medicine utilizing antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) as therapeutic platforms have brought promising solutions for various neurodegenerative/neuromuscular disorders and rare diseases. Currently, 20 oligonucleotide drug products have been commercially approved by the FDA and EMA, and many more are in clinical phase I-III trials. In situ hybridization (ISH) and immunohistochemistry (IHC) are increasingly used to
spatially visualize the delivered therapeutic, target gene, transgene, and/or cell markers and complement information gathered from molecular technologies such as qPCR and digital PCR. Direct visualization of oligonucleotides can also be used to monitor the risk of off-target events by studying biodistribution of potential therapies in various organs.
To expand our current RNAscope™ capabilities on the Roche DISCOVERY ULTRA System, we developed a new ISH assay enabling the detection of ASOs, siRNAs, microRNAs (miRNAs), and other small RNAs (smRNAs) that are 17-50 bases. This fully automated assay allows the co-detection of any combination of smRNA-smRNA, smRNA-mRNA, or mRNA-mRNA, and can be combined with IHC to detect protein targets on the same formalin-fixed paraffin-embedded tissue section. This workflow includes the new protease-free pretreatment reagent VS PretreatPro, allowing for RNA detection without disrupting protease-sensitive epitopes. Visualization of multiple RNAs and proteins at the single-cell level is achieved by combining different HRP and AP-based detections, leveraging Roche’s translucent chromogens.
For proof-of-concept, we used this new automated assay to investigate the expression profile of small RNAs in the mouse brain and combine them with mRNA and protein markers of specific brain cell populations for spatial and morphological context.
This poster was presented at the ASGCT 2025 meeting.
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