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

摘要: RT-qPCR is a common tool for quantification of gene expression, but its accuracy dependent on the choice and stability (steady state expression levels) reference gene/s used normalization. To date, in bone field, there have been few studies to determine most stable genes and, usually, data normalised non-validated genes, commonly GAPDH, ACTB 18 S rRNA. Here we draw attention potential deleterious impact using classical normalise without prior validation their stability. Using geNorm Normfinder programs, panels mouse human were assessed under three different experimental conditions: 1) disease progression Crouzon syndrome (craniosynostosis) model, 2) proliferative culture cranial suture cells isolated from craniosynostosis patients 3) osteogenesis marrow stromal cell line. We demonstrate that are not always ‘stable’ ‘stability’ highly conditions. Selected individually or combination, then osteocalcin alkaline phosphatase during fusion model strategies compared. Strikingly, trends varied significantly when least stable, genes. minimise errors evaluating levels, analysis panel subsequent normalization several strongly recommended over single gene. In particular, conclude use single, “housekeeping” such as rRNA, currently widespread practice by researchers likely produce questionable reliability changes 2 fold less, should be interpreted with due caution.