Abstract
BackgroundAnalysis of gene expression at the mRNA level, using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), mandatorily requires reference genes (RGs) as internal controls. However, increasing evidences have shown that RG expression may vary considerably under experimental conditions. We sought for an appropriate panel of RGs to be used in the 3T3-L1 cell line model during their terminal differentiation into adipocytes. To this end, the expression levels of a panel of seven widely used RG mRNAs were measured by qRT-PCR. The 7 RGs evaluated were ß-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl-transferase I (HPRT), ATP synthase H+ transporting mitochondrial F1 complex beta subunit (ATP-5b), tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein, zeta polypeptide (Ywhaz), Non-POU-domain containing octamer binding protein (NoNo), and large ribosomal protein L13a (RPL).Methodology/Principal FindingsUsing three Excel applications, GeNorm, NormFinder and BestKeeper, we observed that the number and the stability of potential RGs vary significantly during differentiation of 3T3-L1 cells into adipocytes. mRNA expression analyses using qRT-PCR revealed that during the entire differentiation program, only NoNo expression is relatively stable. Moreover, the RG sets that were acceptably stable were different depending on the phase of the overall differentiation process (i.e. mitotic clonal expansion versus the terminal differentiation phase). RPL, ACTB, and Ywhaz, are suitable for terminal differentiation, whereas ATP-5b and HPRT, are suitable during mitotic clonal expansion.ConclusionOur results demonstrate that special attention must be given to the choice of suitable RGs during the various well defined phases of adipogenesis to ensure accurate data analysis and that the use of several RGs is absolutely required. Consequently, our data show for the first time, that during mitotic clonal expansion, the most suitable RGs are ATP-5b, NoNo and HPRT, while during terminal differentiation the most suitable RGs are, NoNo, RPL, ACTB and Ywhaz.
Highlights
Adipose tissue is essentially composed of adipocytes, wherein lipogenesis and lipolysis take place within the frame of energy storage and release, in response to the energy balance status
Selection of Candidate Reference Genes Seven candidates reference genes (RGs) were selected for this study
Due to the extensive use of b-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as RG for gene expression during adipocyte differentiation, they were included in our analysis
Summary
Adipose tissue is essentially composed of adipocytes, wherein lipogenesis and lipolysis take place within the frame of energy storage and release, in response to the energy balance status In addition to their key role in the control of energy metabolism, adipocytes are considered as endocrine cells due to their secretion of adipokines, which are highly influential on, e.g. the immune system, blood vessels and insulin sensitivity [1]. They first reenter the cell cycle, undergo several rounds of division called mitotic clonal expansion (MCE), and undergo the terminal differentiation (TD) [3] These events produce dramatic changes in the expression of a host of genes and proteins [4]. We sought for an appropriate panel of RGs to be used in the 3T3-L1 cell line model during their terminal differentiation into adipocytes To this end, the expression levels of a panel of seven widely used RG mRNAs were measured by qRT-PCR. The 7 RGs evaluated were ß-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl-transferase I (HPRT), ATP synthase H+ transporting mitochondrial F1 complex beta subunit (ATP-5b), tyrosine 3-monooxygenase/tryptophan 5monooxygenase activation protein, zeta polypeptide (Ywhaz), Non-POU-domain containing octamer binding protein (NoNo), and large ribosomal protein L13a (RPL)
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