Housekeeping gene expression variability in differentiating and non-differentiating 3T3-L1 cells

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 (<i>Ppia</i>) and TATA box-binding protein (<i>Tbp</i>) 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.