Abstract: Intrinsic biological fluctuation and/or measurement error can obscure the association of gene expression patterns between RNA and protein levels. Appropriate normalization of reverse-transcription quantitative PCR (RT-qPCR) data can reduce technical noise in transcript measurement, thus uncovering such relationships. The accuracy of gene expression measurement is often challenged in the context of cancer due to the genetic instability and “splicing weakness” involved. Here, we sequenced the poly(A) cancer transcriptome of canine osteosarcoma using mRNA-Seq. Expressed sequences were resolved at the level of two consecutive exons to enable the design of exon-border spanning RT-qPCR assays and ranked for stability based on the coefficient of variation (CV). Using the same template type for RT-qPCR validation, i.e. poly(A) RNA, avoided skewing of stability assessment by circular RNAs (circRNAs) and/or rRNA deregulation. The strength of the relationship between mRNA expression of the tumour marker S100A4 and its proportion score of quantitative immunohistochemistry (qIHC) was introduced as an experimental readout to fine-tune the normalization choice. Together with the essential logit transformation of qIHC scores, this approach reduced the noise of measurement as demonstrated by uncovering a highly significant, strong association between mRNA and protein expressions of S100A4 (Spearman’s coefficient ρ = 0.72 (p = 0.006)). Key messages: • RNA-seq identifies stable pairs of consecutive exons in a heterogeneous tumour. • Poly(A) RNA templates for RT-qPCR avoid bias from circRNA and rRNA deregulation. • HNRNPL is stably expressed across various cancer tissues and osteosarcoma. • Logit transformed qIHC score better associates with mRNA amount. • Quantification of minor S100A4 mRNA species requires poly(A) RNA templates and dPCR.
Bibliographical noteFunding Information:
The Austrian Science Fund financed the NGS data in part ( https://fwf.ac.at/en/ ; project P23336-B11). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was supported by the Genomics Center of the University of Minnesota ( http://genomics.umn.edu ) and by resources and tools of VetCore of the University of Veterinary Medicine Vienna ( www.vetcore.at ). Acknowledgements
© 2020, The Author(s).
Copyright 2020 Elsevier B.V., All rights reserved.
- Quantitative immunohistochemistry
- RNA sequencing
- RT-qPCR data normalization
- Stably consecutive expressed exons
- mRNA-protein correlation
PubMed: MeSH publication types
- Journal Article
- Research Support, Non-U.S. Gov't