Validation of housekeeping genes as an internal control for gene expression studies in the brain of ovariectomized rats treated with tibolone

Introduction: In the central nervous system (CNS), tibolone actions are mainly modulated through its interaction with estrogen, progesterone, and androgen receptors. Several studies have reported the expression of sex hormone receptors in the CNS using the RT-PCR endpoint technique. Although some studies have validated reference genes for rat brain tissue in different experimental conditions, no suitable reference genes have been reported in brain tissue from ovariectomized rats treated with tibolone. Objective: The aim of this investigation was to evaluate the expression of different housekeeping genes in several brain regions in ovariectomized rats treated with tibolone to determine the stability of a single housekeeping gene and a combination of two housekeeping genes under these experimental conditions. Methods: Adult female Sprague-Dawley rats were ovariectomized. Seven days after the surgery, animals were administered a single dose of vehicle (water) or tibolone (10 mg/kg/weight). Twenty-four hours later, animals were sacrificed, and the hypothalamus, hippocampus, prefrontal cortex, and cerebellum were dissected. Total RNA was extracted from these tissues, and RT-qPCR was performed to amplify Ppia, Hprt1, Rpl32, and Gapdh housekeeping genes. Results: Ppia was the most stable gene in the hypothalamus and cerebellum, whereas Hprt1 was the most stable gene in the prefrontal cortex. For the analysis of the combination of two genes, the most stable combination was Ppia and Hrpt1 for the prefrontal cortex and Ppia and Rpl32 for the cerebellum. Conclusion: In ovariectomized rats treated with tibolone, Hprt1 and Ppia genes showed high stability as housekeeping genes for qPCR analysis.

Automated summary

In ovariectomized rats treated with tibolone, Hprt1 and Ppia genes showed high stability as housekeeping genes for qPCR analysis. Ppia was the most stable gene in the hypothalamus and cerebellum, whereas Hprt1 was the most stable gene in the prefrontal cortex. The combination of Ppia and Hprt1 was the most stable for the prefrontal cortex and the combination of Ppia and Rpl32 for the cerebellum.