Selection of Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Three Types of Rat Adipose Tissue

Int J Mol Sci. 2016 Jun 22;17(6):968. doi: 10.3390/ijms17060968.


Quantitative real-time PCR (qRT-PCR) is the most classical technique in the field of gene expression study. This method requires an appropriate reference gene to normalize mRNA levels. In this study, the expression stability of four frequently-used reference genes in epididymal white adipose tissue (eWAT), inguinal beige adipose tissue (iBeAT) and brown adipose tissue (BAT) from obese and lean rats were evaluated by geNorm, NormFinder and BestKeeper. Based on the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, the two most stable reference genes were recommended in each type of adipose tissue. Two target genes were applied to test the stability of the reference genes. The geNorm and NormFinder results revealed that GAPDH and 36B4 exhibited the highest expression stabilities in eWAT, while 36B4 and β-actin had the highest expression stabilities in iBeAT and BAT. According to the results of the BestKeeper analysis, 36B4 was the most stable gene in eWAT, iBeAT and BAT, in terms of the coefficient of variance. In terms of the coefficient of correlation, GAPDH, 36B4 and β-actin were the most stable genes in eWAT, iBeAT and BAT, respectively. Additionally, expected results and statistical significance were obtained using a combination of two suitable reference genes for data normalization. In conclusion, 36B4 and GAPDH, in combination, are the best reference genes for eWAT, while 36B4 and β-actin are two most suitable reference genes for both iBeAT and BAT. We recommend using these reference genes accordingly.

Keywords: BestKeeper; NormFinder; geNorm; rat adipose tissue; reference gene.

MeSH terms

  • Actins / genetics
  • Actins / metabolism
  • Adipose Tissue / metabolism*
  • Animals
  • Gene Expression Profiling / standards*
  • Glyceraldehyde-3-Phosphate Dehydrogenase (NADP+)(Phosphorylating) / genetics
  • Glyceraldehyde-3-Phosphate Dehydrogenase (NADP+)(Phosphorylating) / metabolism
  • Male
  • Rats
  • Rats, Wistar
  • Real-Time Polymerase Chain Reaction / standards*
  • Reference Standards
  • Ribosomal Proteins / genetics
  • Ribosomal Proteins / metabolism


  • Actins
  • Ribosomal Proteins
  • ribosomal protein P0
  • Glyceraldehyde-3-Phosphate Dehydrogenase (NADP+)(Phosphorylating)