RNAscope compatibility with image analysis platforms for the quantification of tissue-based colorectal cancer biomarkers in archival formalin-fixed paraffin-embedded tissue

Arthur E Morley-Bunker; George A R Wiggins; Margaret J Currie; Helen R Morrin; Martin R Whitehead; Tim Eglinton; John Pearson; Logan C Walker

doi:10.1016/j.acthis.2021.151765

RNAscope compatibility with image analysis platforms for the quantification of tissue-based colorectal cancer biomarkers in archival formalin-fixed paraffin-embedded tissue

Acta Histochem. 2021 Sep;123(6):151765. doi: 10.1016/j.acthis.2021.151765. Epub 2021 Aug 5.

Authors

Arthur E Morley-Bunker¹, George A R Wiggins², Margaret J Currie², Helen R Morrin³, Martin R Whitehead⁴, Tim Eglinton⁵, John Pearson², Logan C Walker²

Affiliations

¹ Mackenzie Cancer Research Group, Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand. Electronic address: arthur.morley-bunker@otago.ac.nz.
² Mackenzie Cancer Research Group, Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand.
³ Mackenzie Cancer Research Group, Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand; Cancer Society Tissue Bank, Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand.
⁴ Canterbury Health Laboratories, Christchurch Hospital, Christchurch, New Zealand.
⁵ Department of Surgery, University of Otago, Christchurch, New Zealand.

PMID: 34364165
DOI: 10.1016/j.acthis.2021.151765

Abstract

RNAscope®, has emerged as an important in-situ hybridisation method to validate mRNA expression within single cells whilst preserving tissue morphology in histological samples. The aim of this research was to compare the utility of various open-source and commercial image analysis methods, to quantify mRNA transcripts identified by RNAscope within formalin fixed paraffin embedded (FFPE) histological samples and cell monolayer preparations. Examination of MLH1 expression from 10 histological FFPE colorectal cancer specimens using four image analysis tools (Colour Deconvolution, SpotStudio, WEKA and the LEICA RNA-ISH algorithm) showed the WEKA tool as having the greatest level of agreement with manual quantification. Comparing image analysis methods to qRT-PCR for quantifying MLH1, GFI1 and TNFRSF11A expression within two colorectal cell lines results suggest that these image analysis methods perform at a similar level to qRT-PCR. Furthermore, we describe the strengths and limitations for each image analysis method when used in combination with RNAscope assays. Our study concludes that there are several freely available and commercial image analysis tools that enable reliable RNA in situ expression analysis, however operators need to consider factors, such as expected expression levels of target genes, software usability and functionality.

Keywords: Biomarkers; Colorectal neoplasms; Gene expression; Image analysis; In situ hybridization; RNA; Tumour.

Publication types

Randomized Controlled Trial

MeSH terms

Biomarkers, Tumor / biosynthesis*
Cell Line, Tumor
Colorectal Neoplasms* / metabolism
Colorectal Neoplasms* / pathology
Female
Gene Expression Regulation, Neoplastic*
Humans
Image Processing, Computer-Assisted*
In Situ Hybridization*
Male
Neoplasm Proteins / biosynthesis*
Paraffin Embedding
RNA, Messenger / biosynthesis*
RNA, Neoplasm / biosynthesis*

Substances

Biomarkers, Tumor
Neoplasm Proteins
RNA, Messenger
RNA, Neoplasm