Bone to pick: the importance of evaluating reference genes for RT-qPCR quantification of gene expression in craniosynostosis and bone-related tissues and cells.

BMC Research Notes
Xianxian YangBarry C Powell

Abstract

RT-qPCR is a common tool for quantification of gene expression, but its accuracy is dependent on the choice and stability (steady state expression levels) of the reference gene/s used for normalization. To date, in the bone field, there have been few studies to determine the most stable reference genes and, usually, RT-qPCR data is normalised to non-validated reference genes, most commonly GAPDH, ACTB and 18 S rRNA. Here we draw attention to the potential deleterious impact of using classical reference genes to normalise expression data for bone studies without prior validation of their stability. Using the geNorm and Normfinder programs, panels of mouse and human genes were assessed for their stability under three different experimental conditions: 1) disease progression of Crouzon syndrome (craniosynostosis) in a mouse model, 2) proliferative culture of cranial suture cells isolated from craniosynostosis patients and 3) osteogenesis of a mouse bone marrow stromal cell line. We demonstrate that classical reference genes are not always the most 'stable' genes and that gene 'stability' is highly dependent on experimental conditions. Selected stable genes, individually or in combination, were then used to normalise osteocalcin an...Continue Reading

References

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Citations

Aug 5, 2018·Journal of Cellular Physiology·Rodrigo P F AbunaAdalberto L Rosa
Jan 11, 2014·The Journal of Craniofacial Surgery·Junyi YangXiongzheng Mu
Dec 6, 2017·Journal of Cellular Physiology·Simona CepollaroMilena Fini
Mar 3, 2020·Frontiers in Endocrinology·Robert Brommage, Claes Ohlsson

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Methods Mentioned

BETA
PCR
electrophoresis
protein folding

Software Mentioned

qBasePLUS
RefGenes
Bestkeeper
geNorm
Normfinder

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