Validation of Reference Genes for the Relative Quantification of Gene Expression in Human Epicardial Adipose Tissue

Abstract

Background Relative quantification is a commonly used method for assessing gene expression, however its accuracy and reliability is dependent upon the choice of an optimal endogenous control gene, and such choice cannot be made a priori. There is limited information available on suitable reference genes to be used for studies involving human epicardial adipose tissue. The objective of the current study was to evaluate and identify optimal reference genes for use in the relative quantification of gene expression in human epicardial fat depots of lean, overweight and obese subjects. Methodology/Principal Findings Some of the commonly used reference genes including 18S , ACTB , RPL27 , HPRT , CYCA , GAPDH , RPLPO , POLR2A and B2M were quantified using real-time PCR analysis. The expression stability of these genes was evaluated using Genorm, Normfinder and Bestkeeper algorithms. In addition, the effect of sample size on the validation process was studied by randomly categorizing subjects in two cohorts of n = 2 and n = 33. Conclusions/Significance CYCA , GAPDH and RPL27 were identified as the most stable genes common to all three algorithms and both sample sizes. Their use as reference gene pairs might contribute to the enhanced robustness of relative quantification in the studies involving the human epicardial adipose tissue.

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

“…Two previous studies have demonstrated that GAPDH is a stably expressed gene in human adipose cells across a wide range of experimental settings (14) and in the epicardial adipose tissues of lean, overweight and obese patients undergoing coronary artery bypass grafting (29). Even in studies involving adipocyte differentiation, GAPDH was still seleced as an endogenous reference gene (30,31).…”

Differences between perivascular adipose tissue surrounding the heart and the internal mammary artery: possible role for the leptin-inflammation-fibrosis-hypoxia axis

“…For the selection of an endogenous control gene, a variety of commonly used reference genes as well as genes recommended for AT gene expression studies [25,26] were tested, namely 18S, ACTB, GAPDH, LRP10, PGK1, PPIA, and RPL27. Small-scale analysis with mRNA samples of PVAT depots led to the exclusion of 18S, GAPDH, and RPL27.…”

Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp

“…An internal reference gene or housekeeping gene is often used for this purpose. However, the expression of such genes frequently varies under different conditions, such as physiological state, tissue type, experimental treatments, and species differences (Ullmannova and Haskovec, 2003; Huggett et al , 2005; Ho-pun-Cheung et al , 2008; Infante et al , 2008; Chechi et al , 2012). Therefore, a stable reference gene is to be identified and validated so as to improve the accuracy and reliability of RT-qPCR test.…”