Fully Automated RNAscope In Situ Hybridization Assays for Formalin‐Fixed Paraffin‐Embedded Cells and Tissues. Issue 10 (6th June 2016)

• Record Type: Journal Article
• Authors: Anderson, Courtney M.; Zhang, Bingqing; Miller, Melanie; Butko, Emerald; Wu, Xingyong; Laver, Thomas; Kernag, Casey; Kim, Jeffrey; Luo, Yuling; Lamparski, Henry; Park, Emily; Su, Nan; Ma, Xiao‐Jun
• Is Part Of: Journal of cellular biochemistry. Volume 117:Issue 10(2016:Oct.)
• Page Start: 2201
• Page End: 2208
• Publication Date: 2016-06-06
• Subjects: IN SITU HYBRIDIZATION -- RNA -- RNAscope -- GENE EXPRESSION -- BIOMARKER -- AUTOMATION; Cytochemistry -- Periodicals; 572
• DOI: 10.1002/jcb.25606
• Languages: English
• ISSNs: 0730-2312

Abstract

ABSTRACT: Biomarkers such as DNA, RNA, and protein are powerful tools in clinical diagnostics and therapeutic development for many diseases. Identifying RNA expression at the single cell level within the morphological context by RNA in situ hybridization provides a great deal of information on gene expression changes over conventional techniques that analyze bulk tissue, yet widespread use of this technique in the clinical setting has been hampered by the dearth of automated RNA ISH assays. Here we present an automated version of the RNA ISH technology RNAscope that is adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal‐to‐noise ratio with little to no background staining and results comparable to the manual assay. In addition, the automated duplex RNAscope assay was able to detect two biomarkers simultaneously. Lastly, assay consistency and reproducibility were confirmed by quantification of TATA‐box binding protein ( TBP ) mRNA signals across multiple lots and multiple experiments. Taken together, the data presented in this study demonstrate that the automated RNAscope technology is a high performance RNA ISH assay with broad applicability in biomarker research and diagnostic assay development. J. Cell. Biochem. 117: 2201–2208, 2016. © 2016 Wiley Periodicals, Inc. Abstract : Spatially mapped expression data at the single‐cell level is crucial to understanding cellular organization and cell‐to‐cell interactions in complex tissue. The RNAscope method is a significant advancement in RNA ISH technology that is highly sensitive and specific while providing single‐cell expression resolution with spatial and morphological context. In this study we report the development of an automated RNA ISH assay employing the latest RNAscope technology. This assay generates a high signal to noise ratio and is quantifiable, adaptable to perform on multiple platforms, and capable of duplex gene detection.