Abstract
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.
Highlights
The gradually increasing prevalence of obesity has caused adipose tissue to be a focus
The purity of the isolated RNA from different types of adipose tissue in control and high-fat diet rats was satisfactory for further studies
The specificity of each primer pair was verified by melting curves analysis using a corresponding cDNA sample, only a single quantitative real-time polymerase chain reaction (qRT-PCR) product was amplified (Figure 1C)
Summary
The gradually increasing prevalence of obesity has caused adipose tissue to be a focus. In order to study depot-related differences in mRNA expression in adipose tissue, a sensitive and specific technique, such as quantitative real-time polymerase chain reaction (qRT-PCR) is required [7]. From a survey of different studies of adipose tissue, qRT-PCR data were normalized to different reference genes, such as Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [13], β-actin [14], 18S [15] and acidic ribosomal phosphoprotein P0 (36B4) [16]. No research has studied the stability of frequently-used reference genes in different types of rat adipose tissue. In this study, we used geNorm, NormFinder and BestKeeper to identify four commonly used reference genes in epididymal WAT (eWAT), inguinal beige adipose tissues (iBeAT) and brown adipose tissue (BAT) from obese and lean rats, and to identify the two most suitable reference genes for data normalization. We used two target genes to confirm how the different reference genes affect the final results
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
