Normalization is a crucial step in gene expression analysis to avoid misinterpretation. Reverse transcription-quantitative polymerase chain reaction was used to measure the expression of 10 candidate housekeeping genes in non-differentiated (ND) and differentiated (DI) 3T3-L1 cells on days 5 and 10. We used geNorm, NormFinder, BestKeeper, RefFinder, and the ∆Ct method to evaluate expression stability. The findings revealed that (1) the expression levels of the reference genes changed over time, even in non-differentiating cells, and (2) peptidylprolyl isomerase A ( Ppia ) and TATA box-binding protein ( Tbp ) were stable reference genes for 10 days in both undifferentiated and differentiated 3T3-L1 cells. Notably, the expression of known reference genes in non-differentiating cells was altered throughout the experiment.

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Statins have recently been reported to have anticancer effects and can be used as anticancer agents via drug repositioning. In reverse transcription quantitative polymerase chain reaction (RT-qPCR), the internal reference gene itself must not be affected by any experimental conditions. Since statins are considered to have a wide range of effects on cells via inhibition of the mevalonate pathway, there is a high possibility that statins might vary the expression of internal reference genes, thereby misleading the obtained gene expression data. The present study evaluates the expression stability of the internal reference genes. Statin-sensitive cancer cells (lung cancer-derived HOP-92, prostate cancer-derived PC-3, and melanoma-derived SK-MEL-5 and MDA-MB-435) and statin-resistant cancer cells (lung cancer-derived NCI-H322M and prostate cancer-derived DU-145) were used for the analysis. Atorvastatin was administered at seven different concentrations (0–30 µM). RT-qPCR was performed for the 15 internal reference genes, and then five algorithms were used to evaluate the stability of internal reference genes. Both statin-sensitive and statin-resistant cancer cells showed that atorvastatin affected certain internal reference genes in a dose-dependent manner. The effect of statins on internal reference genes is dependent on the cancer cell type, suggesting that caution should be exercised when comparing expression between cells.