Abstract
In-situ hybridization (ISH) analysis is a highly desirable, versatile approach for assessing biomarker expression status in a spatial context. Most researchers rely on immunostaining (protein targets) or qPCR (mRNA). However, not all proteins can be immunolabeled due to a lack of well-validated antibodies. The qPCR approach, although highly specific, cannot provide spatial information. RNAscope® employs a unique double Z probe that has to bind to the target RNA in tandem in order to be recognized by the preamplifiers and amplifiers. A fluorescent/chromogenic labeled probe then binds to the multiple binding sites of the amplifiers, which improves detection of low expressing RNA and reduces non-specific binding. RNAscope® replaces cumbersome radioactive and chromogenic ISH with more hassle-free chromogen and fluorescence-labelled probes. At the NYULMC Experimental Pathology Core we have integrated RNAscope® with Polaris multispectral imaging and quantitative analysis using different software platforms. About 21 laboratories have used this workflow to address their specific questions. We have also established and validated the newer BaseScope™ assay. In contrast to RNAscope®, which targets lncRNA and mRNA sequences greater than 300nt, BaseScope™ enables detection of short RNA target sequences between 50-300nt. It can be used to detect exon junctions/splice variants, circular RNA, pre-miRNA, and point mutations. We adapted BaseScope™ to co-detect circular RNA and its linear counterpart in a differentiating cell population, which could not be established on glass chamber slides and had to be stained on a plastic petri dish. In conclusion, RNAscope® and BaseScope™ RNA-ISH are powerful alternative strategies for assessing the spatial distribution of critical biomarkers within intact tissues and cells. This approach coupled with sophisticated imaging modalities and downstream analysis support provides new collaborative opportunities for Core aboratories.