ABSTRACT
Objective To observe the change of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression during 3T3-L1 adipocyte differentiation as well as other reference gene expressions.Methods The mRNA expressions of several common reference genes were detected by real time-PCR on day 0,1,3,5,and 7 of 3T3-L1 adipocyte differentiation.Western blot was used to confirm the protein expressions of three common reference genes.Results (1) GAPDH and transferrin receptor(TFRC) mRNA expressions were significantly increased during adipocyte differentiation.GAPDH mRNA level was increased by 5.7,7.6,22.0,and 24.5 folds on day 1,3,5,and 7 after induction of adipocyte differentiation,but no apparent changes of β-actin,α-tubulin,peptidylprolyl isomerase A (PIPA),and 18S mRNA expressions were detected.The expression changes of key transcript factors for adipocyte differentiation such as PPARγ2,C/EBPα,and C/EBPβ were under-estimated by real time-PCR if GAPDH was chosen as the reference gene.Western blotting results showed that the GAPDH protein level increased gradually during adipocyte differentiation,especially on day 5 and 7 after adipocyte differentiation.There were no obvious changes of β-actin and α-tubulin protein expressions.(2) Berberine significantly inhibited mRNA and protein expressions of GAPDH in the process of adipocyte differentiation.GAPDH mRNA levels were reduced by 68.1％ and 66.3％ on day 5 and 7 after induction of adipocyte differentiation,but with no significant change in other reference genes.Conclusion It is not suitable for GAPDH to be used as an endogenous reference gene during 3T3-L1 adipocyte differentiation.