Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples.
BACKGROUND
Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals.


RESULTS
Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes.


CONCLUSIONS
We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.
Mehta
Rohini
R
Molecular and Microbiology Department and Center for the Study of Genomics in Liver Diseases, George Mason University, Fairfax, VA, USA.
Birerdinc
Aybike
A
Hossain
Noreen
N
Afendy
Arian
A
Chandhoke
Vikas
V
Younossi
Zobair
Z
Baranova
Ancha
A
D000199:Actins / Q000235:genetics / Q000378:metabolism / Q000592:standards; D000465:Algorithms; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism / Q000592:standards; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism / Q000592:standards; D050152:Intra-Abdominal Fat / Q000378:metabolism*; D008259:Macroglobulins / Q000235:genetics / Q000378:metabolism / Q000592:standards; D009765:Obesity / Q000235:genetics; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism / Q000592:standards; D012337:RNA, Ribosomal, 18S / Q000235:genetics / Q000378:metabolism / Q000592:standards; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012984:Software; D025822:Ubiquitin C / Q000235:genetics / Q000378:metabolism / Q000592:standards
D000199:Actins; D008259:Macroglobulins; D012337:RNA, Ribosomal, 18S; D025822:Ubiquitin C; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007041:Hypoxanthine Phosphoribosyltransferase; D012319:RNA Polymerase II