Alteration of housekeeping genes and proteins through senescence of human skin fibroblast cells

The expression levels of certain genes or proteins may exhibit various features regardless of the tissue type. The housekeeping genes and reference proteins are often used during the normalization of expression studies. Herein, we aim to determine and validate the most stable reference gene and protein for a healthy human fibroblast cell line. This study used a finite fibroblast cell line derived from healthy human skin (CCD1079Sk). The samples between 25 and until senescence occurs at passage 55 were collected. Morphological changes during aging by cultivation were observed in each passage. Seven genes (ACTB, SDHA, GAPDH, RPLP0, VCL, TBP, 18S) were evaluated during cultivation and ranked according to their stability via the RefFinder interface. This study reports strong variations for both gene and protein expression studies and the morphology of the cells. Notably, during cultivation, the cell morphology showed an irreversible change starting from passage 43, before the known replicative lifespan of CCD1079Sk. The lack of an RPLP0 subunit after passage 39 may limit the cultivation range when protein expression is required. GAPDH is also an ideal reference gene and protein when studies include gene and protein expression. Or, as a second option, Vinculin may be the first choice when mRNA expression is required, and β-Actin is the best choice for reference protein when the studies include only protein expression. Plus, such as SDHA, RPLP0, and TBP should be avoided. These results will benefit further studies, including normalization for both gene, protein, and cell type.