Integration and Validation of RNA ISH, Multispectral Imaging and Analysis Protocols into a Core Environment
| Autor: | Selvaraj S, Valeria Mezzano, Dabovic B, Loomis C |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: | |
| Zdroj: | Scopus-Elsevier Europe PubMed Central |
| Popis: | Identification of biomarkers is a major goal of personalized medicine. Large transcriptome screens have identified new targets and molecular signatures for disease sub-types. However, tissue spatial information, which fundamentally alters in vivo cell behavior and gene expression, is lost. To understand spatial context and validate bulk tissue screens, most researchers rely exclusively on antibodies and immunostaining assays. Unfortunately, this is either not the appropriate choice for some targets, such as long non-coding RNAs, or it is not feasible because no reliable antibodies exist. To address these issues, we have established an alternative work-flow incorporating RNA in situ hybridization (CISH and FISH), whole slide and/or multispectral scanning and image analysis. Methods: RNAscope technology; Leica SCN scanner or Vectra3 multispectral imaging system for image acquisition; ImageJ2/FIJI, R, InForm and Visiopharm software platforms for quantitative analysis. Results: Several laboratories have used this workflow to address their specific questions. For example, we established and validated RNAscope assays for signaling factor transcripts, which are now integrated into an ongoing clinical trial. In this case, all tested commercial antibodies failed the validation assay. We also assessed expression of LNC RNAs in prostate cancer and put in place protocols for normalizing probe quantification across samples. The analysis revealed that storage and/or sample preparation affected the detection of certain LNC RNAs more than others identifying important factors regarding banking specimens. Spatial heat map visualization of RNAscope probes revealed an unexpected distribution of inflammatory cytokine targets in the kidney which are now being further investigated. In conclusion, RNA ISH is a powerful alternative strategy for assessing the spatial distribution of specific cell populations and critical biomarkers within intact tissues. This approach coupled with sophisticated imaging modalities and downstream analysis support provides new collaborative opportunities for Core laboratories. |
| Databáze: | OpenAIRE |
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