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

•RNA in situ expression quantification reliably achieved with image analysis methods.•Image analysis methods perform at similar levels to qRT-PCR.•Consideration to image analysis functionality required for user friendly experience. RNAscope®, has emerged as an important in-situ hybridisation method...

Full description

Saved in:
Bibliographic Details
Published in:Acta histochemica 2021-09, Vol.123 (6), p.151765-151765, Article 151765
Main Authors: Morley-Bunker, Arthur E., Wiggins, George A.R., Currie, Margaret J., Morrin, Helen R., Whitehead, Martin R., Eglinton, Tim, Pearson, John, Walker, Logan C.
Format: Article
Language:English
Subjects:
Biomarkers
Biomarkers, Tumor - biosynthesis
Cell Line, Tumor
Colorectal neoplasms
Colorectal Neoplasms - metabolism
Colorectal Neoplasms - pathology
Female
Gene expression
Gene Expression Regulation, Neoplastic
Humans
Image analysis
Image Processing, Computer-Assisted
In Situ Hybridization
Male
Neoplasm Proteins - biosynthesis
Paraffin Embedding
RNA
RNA, Messenger - biosynthesis
RNA, Neoplasm - biosynthesis
Tumour
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:•RNA in situ expression quantification reliably achieved with image analysis methods.•Image analysis methods perform at similar levels to qRT-PCR.•Consideration to image analysis functionality required for user friendly experience. 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.
ISSN:0065-1281
1618-0372
DOI:10.1016/j.acthis.2021.151765