Reference Gene Validation in the Embryonic and Postnatal Brain in the Rat Hyperhomocysteinemia Model

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.