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

Research output: Contribution to journalArticlepeer-review

  • External authors:
  • Tim Smith
  • Omneya Ahmed Ibrahim Abdelkarem
  • Joely Irlam
  • Brian Lane
  • Helen Valentine
  • Becky Bibby
  • Helen Denley


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, USB, TBP and IPO8 are the most suitable endogenous control genes for bladder cancer qPCR.

Bibliographical metadata

Original languageEnglish
JournalNature -London-
Early online date14 Oct 2020
Publication statusE-pub ahead of print - 14 Oct 2020