Abstract
BackgroundGiven 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.ResultsCross-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.ConclusionsWe 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.
Highlights
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
Genes encoding for 18S, beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-2microglobulin (B2M), hypoxanthine guanine phosphoribosyl transferase1 (HPRT1), tyrosine 3-monooxygensae/ tryptophane 5- monooxygenase activation protein, zeta polypeptide (YWHAZ), ubiquitin C (UBC) and RNA polymerase II (RPII, or POLR2A) were selected according to previously published studies that relied on these genes as reference controls [2,19,11]
GeNorm Analysis Investigation of raw non-normalized data of 5 obese and 4 lean visceral adipose tissue samples (n = 9) allowed sorting of genes ranked on the basis of their expression stability (M) from least stable to most stable (18S T YWHAZ T UBC T B2M T GAPDH T HPRT1 T RPII and ACTB)
Summary
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. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. Accumulation of excessive visceral fat (visceral obesity) is associated with an array of metabolic perturbations including type 2 diabetes, insulin resistance, non-alco-. It has even been suggested that hepatic steatosis itself may be the primary cause of insulin resistance and metabolic syndrome in obesity [13]. Many NAFLD centered studies involve the profiling of adipose samples for the production of various soluble mediators of inflammation produced by components of the visceral fat and released in circulation
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