Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue

Ananya Choudhury,Becky A. S. Bibby,Helen Denley,Tim A. D. Smith,Catharine M. L. West,Brian Lane,Helen Valentine,Joely J. Irlam-Jones,Omneya A. AbdelKarem

doi:10.1038/s41598-020-74380-7

Abstract

Quantitative real time polymerase chain reaction (qPCR) data are normalised using endogenous control genes. We aimed to: (1) demonstrate a pathway to identify endogenous control genes for qPCR analysis of formalin-fixed paraffin-embedded (FFPE) tissue using bladder cancer as an exemplar; and (2) examine the influence of probe length and sample age on PCR amplification and co-expression of candidate genes on apparent expression stability. RNA was extracted from prospective and retrospective samples and subject to qPCR using TaqMan human endogenous control arrays or single tube assays. Gene stability ranking was assessed using coefficient of variation (CoV), GeNorm and NormFinder. Co-expressed genes were identified from The Cancer Genome Atlas (TCGA) using the on-line gene regression analysis tool GRACE. Cycle threshold (Ct) values were lower for prospective (19.49 ± 2.53) vs retrospective (23.8 ± 3.32) tissues (p < 0.001) and shorter vs longer probes. Co-expressed genes ranked as the most stable genes in the TCGA cohort by GeNorm when analysed together but ranked lower when analysed individually omitting co-expressed genes indicating bias. Stability values were < 1.5 for the 20 candidate genes in the prospective cohort. As they consistently ranked in the top ten by CoV, GeNorm and Normfinder, UBC, RPLP0, HMBS, GUSB, and TBP are the most suitable endogenous control genes for bladder cancer qPCR.

Highlights

Quantitative real time polymerase chain reaction data are normalised using endogenous control genes
Gene expression was determined in triplicate and the intra-sample standard deviation (SD) of the cycle threshold (Ct) values calculated for the 28 samples
To investigate inter-assay reliability three samples were assayed by Quantitative real time polymerase chain reaction (qPCR) on two separate TaqMan arrays/96 well plates run on two different days

Summary

Introduction

Quantitative real time polymerase chain reaction (qPCR) data are normalised using endogenous control genes. We aimed to: (1) demonstrate a pathway to identify endogenous control genes for qPCR analysis of formalin-fixed paraffin-embedded (FFPE) tissue using bladder cancer as an exemplar; and (2) examine the influence of probe length and sample age on PCR amplification and co-expression of candidate genes on apparent expression stability. Stability values were < 1.5 for the 20 candidate genes in the prospective cohort As they consistently ranked in the top ten by CoV, GeNorm and Normfinder, UBC, RPLP0, HMBS, GUSB, and TBP are the most suitable endogenous control genes for bladder cancer qPCR. Test data generated are subject to stability assessment algorithms, the two most commonly used are GeNorm[11] and NormFinder[12] These algorithms rank genes in order of stability and in the case of GeNorm select the two-gene combination that provides the most stable normalization. GeNorm is considered the optimal algorithm for studies with small sample n umbers[12] but over-rates the stability of co-expressed genes in the candidate panel

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