Lateralization of housekeeping genes in the brain of one-day old chicks

Real-time quantitative PCR is an exceptionally sensitive method that can detect even very small differences in gene expression and, as such, it is essential to use suitable reference genes. Domestic chickens are used in a wide range of studies including neurobiology, behavior, ecology and disease transmission. In recent avian gene expression experiments, 18S (18S ribosomal RNA), beta actin (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have frequently been used; however, there is not enough evidence that these reference genes are suitable for all types of experiments. There is considerable evidence for lateralization in numerous learning tasks and for differences in the functional contribution of the two brain hemispheres. Therefore, the purpose of this study was to identify a set of reference genes for chick brain region called an intermediate medial mesopallium (IMM), which is connected with memory formation in the chick brain, whilst also taking into consideration the differences between the left and right hemispheres. This study evaluated the expression stability of eleven candidate housekeeping genes in the IMM region of the 1-day old chick brain. In our experimental system, the most reliable results were given by the NormFinder algorithm. The results show for the first time that ACTB, commonly used as an avian reference gene, is not suitable for investigation of gene expression in the chick brain and that brain lateralization exact selection of different reference gens for each hemisphere. For memory process investigations using tasks in one-day old chicks the most effective reference genes for the left hemisphere were HMBS and SDHA, and for the right hemisphere the most effective was RPL19.