Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation

  • Authors:
    • Juan Zhang
    • Hongju Tang
    • Yuqing Zhang
    • Ruyuan Deng
    • Li Shao
    • Yun Liu
    • Fengying Li
    • Xiao Wang
    • Libin Zhou
  • View Affiliations

  • Published online on: March 11, 2014     https://doi.org/10.3892/ijmm.2014.1695
  • Pages: 1209-1218
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important in the effort to gain insight into the molecular mechanisms underlying adipogenesis. However, the expression profile of a target gene may be misinterpreted due to the unstable expression of the reference genes under different experimental conditions. Therefore, in this study, we investigated the expression stability of 10 commonly used reference genes during 3T3-L1 adipocyte differentiation. The mRNA expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferrin receptor (TFRC) significantly increased during the course of 3T3-L1 adipocyte differentiation, which was decreased by berberine, an inhibitor of adipogenesis. Three popular algorithms, GeNorm, NormFinder and BestKeeper, identified 18 ribosomal RNA and hydroxymethylbilane synthase (HMBS) as the most stable reference genes, while GAPDH and TFRC were the least stable ones. Peptidylprolyl isomerase A [PIPA (cyclophilin A)], ribosomal protein, large, P0 (36-B4), beta-2-microglobulin (B2M), α1-tubulin, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β-actin showed relatively stable expression levels. The choice of reference genes with various expression stabilities exerted a profound influence on the expression profiles of 2 target genes, peroxisome proliferator-activated receptor (PPAR)γ2 and C/EBPα. In addition, western blot analysis revealed that the increased protein expression of GAPDH was markedly inhibited by berberine during adipocyte differentiation. This study highlights the importance of selecting suitable reference genes for qRT-PCR studies of gene expression during the process of adipogenesis.
View Figures
View References

Related Articles

Journal Cover

May-2014
Volume 33 Issue 5

Print ISSN: 1107-3756
Online ISSN:1791-244X

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Zhang J, Tang H, Zhang Y, Deng R, Shao L, Liu Y, Li F, Wang X and Zhou L: Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation. Int J Mol Med 33: 1209-1218, 2014
APA
Zhang, J., Tang, H., Zhang, Y., Deng, R., Shao, L., Liu, Y. ... Zhou, L. (2014). Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation. International Journal of Molecular Medicine, 33, 1209-1218. https://doi.org/10.3892/ijmm.2014.1695
MLA
Zhang, J., Tang, H., Zhang, Y., Deng, R., Shao, L., Liu, Y., Li, F., Wang, X., Zhou, L."Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation". International Journal of Molecular Medicine 33.5 (2014): 1209-1218.
Chicago
Zhang, J., Tang, H., Zhang, Y., Deng, R., Shao, L., Liu, Y., Li, F., Wang, X., Zhou, L."Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation". International Journal of Molecular Medicine 33, no. 5 (2014): 1209-1218. https://doi.org/10.3892/ijmm.2014.1695