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, Jacqueline James

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Abstract

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.
Original languageEnglish
Number of pages12
JournalOncotarget
Early online date16 Oct 2017
DOIs
Publication statusEarly online date - 16 Oct 2017

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Paraffin
Formaldehyde
In Situ Hybridization
Messenger RNA
Neoplasms
Biomarkers
Immunohistochemistry
Technology
Sensitivity and Specificity
Antibodies
Research

Cite this

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title = "RNAscope in situ hybridization confirms mRNA integrity in formalin-fixed, paraffin-embedded cancer tissue samples",
abstract = "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.",
author = "Victoria Bingham and Leanne McIlreavey and Christine Greene and Edwina O'Doherty and Rebecca Clarke and Stephanie Craig and Manuel Salto-Tellez and Stephen McQuaid and Claire Lewis and Jacqueline James",
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T1 - RNAscope in situ hybridization confirms mRNA integrity in formalin-fixed, paraffin-embedded cancer tissue samples

AU - Bingham, Victoria

AU - McIlreavey, Leanne

AU - Greene, Christine

AU - O'Doherty, Edwina

AU - Clarke, Rebecca

AU - Craig, Stephanie

AU - Salto-Tellez, Manuel

AU - McQuaid, Stephen

AU - Lewis, Claire

AU - James, Jacqueline

PY - 2017/10/16

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N2 - 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.

AB - 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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