使用Roche DISCOVERY™ULTRA在肿瘤组织中自动化共检测小RNA、RNA和蛋白质

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. The FDA has issued recommendations to generate nonclinical biodistribution (BD) data for gene therapy products to evaluate and interpret nonclinical pharmacology and toxicology findings before initiating human clinical trials. I n 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 quantitative polymerase chain reaction (qPCR) and digital PCR. Direct visualization of oligonucleotides can also monitor the risk of off-target events by studying BD of potential therapies in various organs. A new RNAscope™ ISH assay enabling the detection of ASOs, siRNAs, microRNAs (miRNAs), and other small RNAs was developed on the Roche DISCOVERY™ ULTRA System. This fully automated assay allows the co-detection of small RNAs, RNAs and proteins within the same formalin-fixed paraffin-embedded sections of tissues. This workflow includes protease-free RNA detection to prevent disruption of protease-sensitive epitopes. Multiple RNA species and protein targets can be visualized either chromogenically or fluorescently at the single-cell level, leveraging translucent chromogens or TSA-fluorophores. We investigated the expression profile of miR-21, a microRNA implicated in cancer proliferation and progression, in the human cancer TMA tissue. MicroRNA detection was combined with PTEN RNA to assess the impact of miR21 on the expression of PTEN , a tumor suppressor that regulates cell growth, proliferation, and survival. Ki67, and CD31 proteins were co-detected in the same sample to correlate with tumor cell proliferation, and neo-angiogenesis, respectively. This new automated ISH assay showed high sensitivity and specificity for the detection of small RNAs with different expression profiles, and subcellular resolution in intact tissue context. This novel assay will be particularly valuable for the study of ASO/siRNAs delivery, biodistribution, stability, and expression profile of their associated RNA targets for the development of new oligonucleotide therapeutics.