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

Abstract

The 'gold standard' for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current 'gold standard' method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.

Absence of Cytomegalovirus in Glioblastoma and Other High-grade Gliomas by Real-time PCR, Immunohistochemistry, and In Situ Hybridization

“…Improvements in assay specificity results from the requirement for simultaneous hybridization of two separate but adjacent probes for signal detection, and assay sensitivity results from amplification of the signals based on both branching DNA hybridizations and enzymatic reactions. The Advanced Cell Diagnostics (ACD) commercial CISH assay can detect a single DNA or RNA molecule in individual cells (25, 27, 31). Moreover, almost half of our prospective population was CMV seronegative, and it would be highly unlikely that relatively high tissue levels of CMV would be present without evidence of prior infection by serum immunological measures such as the one used here.…”

Long Noncoding RNA Discovery in Cardiovascular Disease

“…47 Another alternative is RNAscope—a novel multiplex nucleic acid in situ hybridization technology that has been used to image lncRNAs. 50 One caveat relevant to the application of these techniques in cardiovascular disease is that successful imaging can be highly cell-type dependent. Therefore, judicious titration of fixation settings, protease treatment, and hybridization conditions is often required.…”

Expression Patterns of Ciliopathy Genes ARL3 and CEP120 Reveal Roles in Multisystem Development

“…Samples were then prepared for the RNAscope assay, a RNA in situ detection platform for detection of target RNA within intact cells as per manufacturers' instructions (33,34). An RNAscope 2.5 Assay RED was employed with 20 paired probes across nucleotides 169-1570 (NM_004311.3) and 115-1133 (NM_001166226.1) for detection of ARL3 and CEP120, respectively and counterstained with Methyl Green.…”