A GeNorm algorithm-based selection of reference genes for quantitative real-time PCR in skin biopsies of healthy dogs and dogs with atopic dermatitis

Quantitative real time PCR (Q-PCR) is the method of choice to study mRNA expression levels. Since Q-PCR is very sensitive, normalization of the data with stably expressed reference genes if of utmost importance. The stability of reference genes depends on the tissue and the species of interest. Therefore, evaluation of the stability of reference genes must be performed for each new tissue and species under study. The stability of B2M, GAPDH, HPRT, SRPR, hnRNPH, GUSB, RPL8, RPS5, and RPS19 was analyzed with the GeNorm software in snap frozen canine skin biopsies. Healthy dogs (n = 7) and dogs with confirmed atopic dermatitis (n = 28) were included. Lesional and non-lesional skin was analyzed. The study indicated that the most appropriate reference genes in canine skin are the ribosomal gene products RPL8, RPS5 and RPS19 besides GUSB and HPRT. As little as three reference genes will reveal highly reliable Q-PCR calculations.