Evaluation of candidate reference genes for quantitative real-time PCR normalization in blood from red deer developing antlers.

Sexual selection favors male traits that increase their ability to monopolize the breeding access to several females. Deer antlers are cranial appendages that regenerate annually in males. Throughout life, the phenology of antler growth advances and antler mass increases until the stag reaches, between 8 and 10 years old, maximum body mass and highest reproductive success. The molecular mechanisms of antler development are of great interest in both evolutionary and regenerative medicine studies. To minimize errors in the assessment of gene expression levels by qRT-PCR, we analyzed the stability of a panel of eight candidate reference genes and concluded that qRT-PCR normalization to three stable genes is strongly convenient in experiments performed in red deer antler blood. To validate our proposal, we compared the expression level of three genes linked to red deer antler growth (ANXA2, APOD and TPM1) in fifteen male red deer classified as young (up to 4 years old) and adults (4-6 years old). Our data confirms that B2M, ACTB and RPLP0 are valuable reference genes for future gene expression studies in red deer antler blood, which would provide increased insight into the effects of intrinsic factors that determine antler development in red deer.