RNAscope

Abstract

In situ analysis of biomarkers is highly desirable in molecular pathology because it allows the examination of biomarker status within the histopathological context of clinical specimens. Immunohistochemistry and DNA in situ hybridization (ISH) are widely used in clinical settings to assess protein and DNA biomarkers, respectively, but clinical use of in situ RNA analysis is rare. This disparity is especially notable when considering the abundance of RNA biomarkers discovered through whole-genome expression profiling. This is largely due to the high degree of technical complexity and insufficient sensitivity and specificity of current RNA ISH techniques. Here, we describe RNAscope, a novel RNA ISH technology with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. RNAscope is compatible with routine formalin-fixed, paraffin-embedded tissue specimens and can use either conventional chromogenic dyes for bright-field microscopy or fluorescent dyes for multiplex analysis. Unlike grind-and-bind RNA analysis methods such as real-time RT-PCR, RNAscope brings the benefits of in situ analysis to RNA biomarkers and may enable rapid development of RNA ISH-based molecular diagnostic assays.

Inhibition of Nox4‐dependent ROS signaling attenuates prostate fibroblast activation and abrogates stromal‐mediated protumorigenic interactions

“…As Nox4 is constitutively active and primarily regulated at the mRNA level,15, 16 we utilized in situ hybridization (ISH) on prostate tissue sections and a PCa tissue microarray (TMA) to identify the cellular origin and semi‐quantify prostatic Nox4 mRNA levels using a highly specific assay with single mRNA transcript sensitivity (Supporting Information, Fig. S1) 29. Nox4 mRNA was detected at low levels in both epithelial and stromal cells of the benign prostate (0.07% of cells in benign areas showed 1–2 mRNA transcript signals).…”

E2F1 controls germ cell apoptosis during the first wave of spermatogenesis

“…ACD‐310035, Advanced Cell Diagnostics, Hayward, CA, USA) was used to detect E2f1 mRNA transcripts on PFA‐fixed, paraffin embedded section from 6, 10, 20, and 40‐day‐old wild‐type testis (Wang et al ., 2012). The probe against mmE2f1 was custom‐made and the standard positive control (Mm‐PPIB, cat.…”

RNAscope for <em>In situ</em> Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

“…However, detection of E6/E7 mRNA from fresh tumor tissue by RT-PCR technique is not practical in routine clinical practice and has been limited to the research laboratories. Recently, we have developed a novel RNA ISH technology called RNAscope, which enables multiplex detection in individual cells with single RNA molecule sensitivity in formalin fixed paraffin embedded tissue specimens (FFPE) [5][6][7][8][9][10] . We provided three lines of evidence for single molecule detection 5 .…”

“…Recently, we have developed a novel RNA ISH technology called RNAscope, which enables multiplex detection in individual cells with single RNA molecule sensitivity in formalin fixed paraffin embedded tissue specimens (FFPE) [5][6][7][8][9][10] . We provided three lines of evidence for single molecule detection 5 . First, the RNAscope probe design and signal amplification system allowed detection of single copy HER2 genomic DNA targets in HeLa and SK-BR-3 cell lines.…”