Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue
Authors
Smith, Tim A DAbdelKarem, Omneya A
Irlam-Jones, Joely J
Lane, Brian
Valentine, Helen R
Bibby, Becky A
Denley, H.
Choudhury, Ananya
West, Catharine M L
Affiliation
Translational Radiobiology Group, Division of Cancer Sciences, University of Manchester, Manchester Academic Health Centre, Christie Hospital NHS Found Trust, Manchester, M20 4BX, UK.Issue Date
2020
Metadata
Show full item recordAbstract
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.Citation
Smith TAD, AbdelKarem OA, Irlam-Jones JJ, Lane B, Valentine H, Bibby BAS, et al. Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue. Sci Rep. 2020;10(1):17258.Journal
Scientific ReportsDOI
10.1038/s41598-020-74380-7PubMed ID
33057113Additional Links
https://dx.doi.org/10.1038/s41598-020-74380-7Type
ArticleLanguage
enae974a485f413a2113503eed53cd6c53
10.1038/s41598-020-74380-7