Research Papers: Pathology:

RNAscope in situ hybridization confirms mRNA integrity in formalin-fixed, paraffin-embedded cancer tissue samples

Victoria Bingham, Leanne McIlreavey, Christine Greene, Edwina O’Doherty, Rebecca Clarke, Stephanie Craig, Manuel Salto-Tellez, Stephen McQuaid _, Claire Lewis and Jacqueline James

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Oncotarget. 2017; 8:93392-93403. https://doi.org/10.18632/oncotarget.21851

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Victoria Bingham2, Leanne McIlreavey2, Christine Greene1,3, Edwina O’Doherty1,3, Rebecca Clarke2, Stephanie Craig2, Manuel Salto-Tellez2, Stephen McQuaid1,2,3 Claire Lewis1,2 and Jacqueline James1,2,3

1 Northern Ireland Biobank, Centre for Cancer Research and Cell Biology, Queen’s University, Belfast, UK

2 Molecular Pathology Programme, Centre for Cancer Research and Cell Biology, Queen’s University, Belfast, UK

3 Tissue Pathology, Belfast Health and Social Care Trust, Belfast City Hospital, Belfast, UK

Correspondence to:

Stephen McQuaid, email:

Keywords: mRNA; FFPE; in situ hybridization; Integrity; Pathology Section

Received: July 27, 2017 Accepted: October 05, 2017 Published: October 16, 2017


Immunohistochemistry remains the overwhelming technique of choice for test biomarker evaluation in both clinical or research settings when using formalin-fixed, paraffin embedded tissue sections. However, validations can be complex with significant issues about specificity, sensitivity and reproducibility. The vast array of commercially available antibodies from many vendors may also lead to non-standard approaches which are difficult to cross-reference. In contrast mRNA detection, by in situ hybridization (ISH) with sequence specific probes, offers a realistic alternative, with less validation steps and more stringent and reproducible assessment criteria. In the present study mRNA ISH was evaluated in prospectively and retrospectively collected FFPE samples within a cancer biobank setting. Three positive control probes, POLR2A, PPIB and UBC were applied to FFPE sections from a range of tumour types in FFPE whole-face (prospective collection) or TMA (retrospective collection) formats and evaluated semi-quantitatively and by image analysis. Results indicate that mRNA can be robustly evaluated by ISH in prospectively and retrospectively collected tissue samples. Furthermore, for 2 important test biomarkers, PD-L1 and c-MET, we show that mRNA ISH is a technology that can be applied with confidence in the majority of tissue samples because there are quantifiable levels of control probes indicating overall mRNA integrity.

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