Lenart Jacek, Kogut Klaudia, Salinska Elzbieta
Department of Neurochemistry, Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, Poland.
Department of Neurochemistry, Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, Poland.
Gene Expr Patterns. 2017 Nov;25-26:85-91. doi: 10.1016/j.gep.2017.06.006. Epub 2017 Jun 15.
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.
实时定量PCR是一种极其灵敏的方法，甚至能够检测出基因表达中非常微小的差异，因此，使用合适的内参基因至关重要。家鸡被广泛应用于包括神经生物学、行为学、生态学和疾病传播等众多研究中。在最近的禽类基因表达实验中，18S（18S核糖体RNA）、β-肌动蛋白（ACTB）和甘油醛-3-磷酸脱氢酶（GAPDH）经常被使用；然而，没有足够的证据表明这些内参基因适用于所有类型的实验。有大量证据表明，在众多学习任务中存在脑侧化现象，并且两个脑半球的功能贡献存在差异。因此，本研究的目的是为鸡脑一个名为中内侧中脑皮质（IMM）的区域确定一组内参基因，该区域与鸡脑的记忆形成有关，同时还要考虑左右半球之间的差异。本研究评估了11个候选管家基因在1日龄雏鸡脑IMM区域的表达稳定性。在我们的实验系统中，NormFinder算法给出了最可靠的结果。结果首次表明，通常用作禽类内参基因的ACTB不适用于雏鸡脑基因表达的研究，并且脑侧化需要为每个半球精确选择不同的内参基因。对于使用1日龄雏鸡进行任务的记忆过程研究，左半球最有效的内参基因是HMBS和SDHA，右半球最有效的是RPL19。
