Maternal hyperhomocysteinemia (HCY) induced by genetic defects in methionine cycle enzymes or vitamin imbalance is known to be a pathologic factor that can impair embryonal brain development and cause long-term consequences in the postnatal brain development as well as changes in the expression of neuronal genes. Studies of the gene expression on this model requires the selection of optimal housekeeping genes. This work aimed to analyze the expression stability of housekeeping genes in offspring brain. Pregnant female Wistar rats were treated daily with a 0.15% L-methionine solution in the period starting on the 4th day of pregnancy until delivery, to cause the increase in the homocysteine level in fetus blood and brain. Housekeeping gene expression was assessed by RT-qPCR on whole embryonic brain and selected rat brain areas at P20 and P90. The amplification curves were analyzed, and raw means Cq data were imported to the RefFinder online tool to assess the reference genes stability. Most of the analyzed genes showed high stability of mRNA expression in the fetal brain at both periods of analysis (E14 and E20). However, the most stably expressed genes at different age points differed. Actb, Ppia, Rpl13a are the most stably expressed on E14, Ywhaz, Pgk1, Hprt1 – on E20 and P20, Hprt1, Actb, and Pgk1 – on P90. Gapdh gene used as a reference in various studies demonstrates high stability only in the hippocampus and cannot be recommended as the optimal reference gene on HCY model. Hprt1 and Pgk1 genes were found to be the most stably expressed in the brain of rat subjected to HCY. These two genes showed high stability in the brain on E20 and in various areas of the brain on the P20 and P90. On E14, the preferred genes for normalization are Actb, Ppia, Rpl13a.

由蛋氨酸循环酶遗传缺陷或维生素失衡引起的母体高同型半胱氨酸血症（HCY）已知是一种病理因素，会损害胚胎大脑发育，对出生后大脑发育造成长期后果以及神经元基因表达的变化。该模型上基因表达的研究需要选择最佳的管家基因。本工作旨在分析子代大脑中管家基因的表达稳定性。妊娠雌性Wistar大鼠在妊娠第4天至分娩期间每天用0.15%L-蛋氨酸溶液处理，引起胎儿血液和脑中同型半胱氨酸水平升高。通过 RT-qPCR 评估整个胚胎大脑和 P20 和 P90 时选定的大鼠大脑区域的管家基因表达。分析扩增曲线，并将原始均值 Cq 数据导入 RefFinder 在线工具以评估内参基因的稳定性。大多数分析的基因在两个分析阶段（E14 和 E20）都显示出胎儿大脑中 mRNA 表达的高度稳定性。然而，不同年龄点最稳定表达的基因有所不同。Actb、Ppia、Rpl13a在 E14 上表达最稳定，Ywhaz、Pgk1、Hprt1在 E20 上和 P20 上表达最稳定，Hprt1、Actb和Pgk1在 P90 上表达。在各种研究中用作参考的Gapdh基因仅在海马中表现出高稳定性，不能推荐作为 HCY 模型的最佳参考基因。发现Hprt1和Pgk1基因在接受 HCY 的大鼠大脑中表达最稳定。这两个基因在 E20 的大脑中以及 P20 和 P90 的大脑各个区域中表现出高度稳定性。在 E14 上，标准化的首选基因是Actb、Ppia、Rpl13a。