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Reference Gene Validation in the Embryonic and Postnatal Brain in the Rat Hyperhomocysteinemia Model
Anna A Kovalenko1, Alexander P Schwarz1, Anastasiia D Shcherbitskaia1,2
1I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences, St. Petersburg, Russia.
Insights
Maternal hyperhomocysteinemia (HCY) impairs fetal brain development. This study identified the most stable housekeeping genes for accurate gene expression analysis in offspring brains affected by HCY.
Area of Science:
- Neuroscience
- Developmental Biology
- Genetics
Background:
- Maternal hyperhomocysteinemia (HCY) is linked to impaired embryonic brain development and long-term neurological consequences.
- Accurate gene expression studies in HCY models require stable housekeeping genes for normalization.
- Previous studies lack comprehensive analysis of housekeeping gene stability across different developmental stages in HCY-affected offspring brains.
Purpose of the Study:
- To evaluate the expression stability of commonly used housekeeping genes in the brains of rat offspring exposed to maternal HCY.
- To identify the most reliable reference genes for accurate gene expression analysis in this specific developmental neurotoxicity model.
Main Methods:
- Pregnant Wistar rats were administered L-methionine to induce HCY, affecting fetal blood and brain homocysteine levels.
- Housekeeping gene expression was assessed using RT-qPCR in whole embryonic brains and specific brain regions at embryonic day 14 (E14), E20, postnatal day 20 (P20), and P90.
- Gene expression stability was analyzed using the RefFinder online tool.
Main Results:
- Most tested housekeeping genes exhibited stable mRNA expression in fetal brains at E14 and E20.
- Optimal housekeeping genes varied by developmental stage: Actb, Ppia, Rpl13a at E14; Ywhaz, Pgk1, Hprt1 at E20 and P20; Hprt1, Actb, Pgk1 at P90.
- Gapdh showed limited stability, only in the hippocampus, and is not recommended as a universal reference gene for HCY models.
- Hprt1 and Pgk1 demonstrated the highest overall stability across multiple brain regions and developmental time points (E20, P20, P90).
Conclusions:
- The selection of appropriate housekeeping genes is crucial for reliable gene expression studies in maternal hyperhomocysteinemia models.
- Hprt1 and Pgk1 are recommended as the most stable reference genes for analyzing gene expression in the brains of HCY-affected rats from embryonic to adult stages.
- Actb, Ppia, and Rpl13a are suitable for normalization at the embryonic day 14 stage.
Abstract:
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.
