Selection of an inadequate housekeeping gene leads to misinterpretation of target gene expression in zinc deficiency and zinc supplementation models

Background: Numerous studies try to find the most stable housekeeping gene for a specific experimental setup. However, there is no universal housekeeping gene described so far and, therefore, new testing of housekeeping genes at the beginning of a new experiment is of high importance. Methods: In the present study, target gene expression of mitochondrial serine/threonine-protein phosphatase (PGAM)5, nuclear factor erythroid 2-related factor (Nrf)2, dynamin related protein (Drp)1 and kelch like ECH associated protein (Keap)1 was tested in zinc-deficient and zinc-supplemented THP-1 cells and compared to control cells. Normalization of results obtained by quantitative real-time polymerase chain reaction (qRT-PCR) was performed by using the housekeeping genes porphobilinogen deaminase (PBGD), beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Additionally, this 3 housekeeping genes were tested in Jurkat cells under these conditions. Results: Surprisingly, analyses of one and the same target gene revealed opposite results depending on the used housekeeping gene. This was caused by significant altered housekeeping gene expressions due to zinc availability. Conclusion: Therefore, this study highlights the importance of choosing adequate housekeeping genes, which might comprise the use of more than one housekeeping gene when different conditions are tested, such as zinc deficiency and zinc supplementation. Related to the herein used experimental setup, the use ofGAPDH to study gene expression upon zinc deficiency and PBGD to study gene expression after zinc supplementation is recommended.