Identification of qPCR reference genes suitable for normalising gene expression in the developing mouse embryo.

Background : Progression through mammalian embryogenesis involves many interacting cell types and multiple differentiating cell lineages. Quantitative polymerase chain reaction (qPCR) analysis of gene expression in the developing embryo is a valuable tool for deciphering these processes, but normalisation to stably-expressed reference genes is essential for such analyses. Gene expression patterns change globally and dramatically as embryonic development proceeds, rendering identification of consistently appropriate reference genes challenging. Methods : We have investigated expression stability in mouse embryos from mid to late gestation (E11.5-E18.5), both at the whole-embryo level, and within the head and forelimb specifically, using 15 candidate reference genes ( ACTB, 18S, SDHA, GAPDH, HTATSF1, CDC40, RPL13A, CSNK2A2, AP3D1, HPRT1, CYC1, EIF4A, UBC, B2M and PAK1IP1 ), and four complementary algorithms (geNorm, Normfinder, Bestkeeper and deltaCt). Results : Unexpectedly, all methods suggest that many genes within our candidate panel are acceptable references, though AP3D1 , RPL13A and PAK1IP1 are the strongest performing genes overall. HPRT1 and B2M are conversely poor choices, and show strong developmental regulation. We further show that normalisation using our three highest-scoring references can reveal subtle patterns of developmental expression even in genes ostensibly ranked as acceptably stable ( CDC40 , HTATSF1 ). Conclusion : AP3D1 , RPL13A and PAK1IP1 represent universally suitable reference genes for expression studies in the E11.5-E18.5 mouse embryo.