A distinctive point of our study is the major effort put into primer design with the aim to validate only oligos spanning at least one intron. This aspect has been neglected in the previous works on reference gene validation in sheep, probably because of the lack of ovine genomic DNA sequences available in the public databases. Indeed, we were able to retrieve intron-spanning primers from previous publications for only three genes (PGK1, SDHA and G6PD) among those included in our study. Nevertheless, this approach is highly recommended in combination with DNAse I treatment to avoid/recognize co-amplification of contaminating genomic DNA [1] , since spurious PCR signals could affect the selection of reliable references by mimicking individual variation with lower stability scores. Also, when searching RTPrimerDB, a reference database for qRT-PCR primers [27] [28] [29] [30] , we noted that among 8329 real-time PCR primer sets for 5758 genes of 26 organisms available at the time of writing, only 16 SYBR Green assays were deposited under Ovis aries. Importantly, therefore, an additional outcome of our study is a set of validated primer sequences suitable for gene expression experiments based on SYBR Green chemistry to be carried out with other ovine tissues and cells.