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Optimal Reference Genes for RT-qPCR Experiments in Hippocampus and Cortex of Rats Chronically Exposed to Excessive
Olga V Nadei1, Natalia I Agalakova2
1Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 194223, Saint-Petersburg, Russia.
Biological Trace Element Research
|April 3, 2023
Summary
Accurate gene expression analysis in rat brain exposed to fluoride requires validated reference genes. This study identified optimal reference genes for quantitative real-time PCR (RT-qPCR) in cortex and hippocampus, improving fluoride toxicity research.
Area of Science:
- Neuroscience
- Toxicology
- Molecular Biology
Background:
- Quantitative real-time PCR (RT-qPCR) relies on stable reference genes for accurate data normalization.
- Previous studies on fluoride toxicity in brain tissues used non-validated reference genes, potentially leading to unreliable results.
Purpose of the Study:
- To identify and validate the most stable reference genes for RT-qPCR analysis in rat cortex and hippocampus following chronic excessive fluoride exposure.
- To provide reliable normalization strategies for gene expression studies in fluoride-induced neurotoxicity.
Main Methods:
- Six candidate reference genes (Gapdh, Pgk1, Eef1a1, Ppia, Tbp, Helz) were analyzed in rat brain tissues (cortex and hippocampus) after 12 months of exposure to varying fluoride concentrations (0.4, 5, 20, 50 ppm).
- Gene expression stability was assessed using coefficient of variation (CV) analysis and the RefFinder program, integrating Delta-Ct, BestKeeper, NormFinder, and GeNorm statistical methods.
Main Results:
- Pgk1, Eef1a1, and Ppia demonstrated the highest stability in the cortex.
- Ppia, Eef1a1, and Helz were the most stable in the hippocampus.
- Tbp and Helz were identified as the least stable in the cortex, while Gapdh and Tbp were unsuitable for the hippocampus.
Conclusions:
- The geometric mean of Pgk1 and Eef1a1 is recommended for normalizing RT-qPCR data in the rat cortex under chronic fluoride exposure.
- The geometric mean of Ppia and Eef1a1 is recommended for normalizing RT-qPCR data in the rat hippocampus under chronic fluoride exposure.
- Validated reference genes are crucial for accurate mRNA quantification in fluoride neurotoxicity studies.
