Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue
AuthorsSmith, Tim A. D.; email: firstname.lastname@example.org
AbdelKarem, Omneya A.
Irlam-Jones, Joely J.
Bibby, Becky A. S.
West, Catharine M. L.
MetadataShow full item record
AbstractAbstract: 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.
CitationScientific Reports, volume 10, issue 1, page 17258
PublisherNature Publishing Group UK
DescriptionFrom Springer Nature via Jisc Publications Router
History: received 2020-06-16, accepted 2020-09-28, registration 2020-10-01, online 2020-10-14, pub-electronic 2020-10-14, collection 2020-12
Publication status: Published
Funder: Mission Sector of the Egyptian Ministry of Higher Education and Scientific Research.
Funder: CRUK; Grant(s): C147/A25254