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

Kanta Chechi,Yves Gelinas,Patrick Mathieu,Denis Richard,Yves Deshaies,Baochuan Lin

doi:10.1371/journal.pone.0032265

Abstract

BackgroundRelative 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 FindingsSome 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.

Highlights

Human epicardial adipose tissue (EAT) is a visceral fat depot that has gained significant attention in the recent times
Relative quantification of gene expression remains the method of choice, its accuracy and reliability is critically dependent upon the choice of endogenous control or housekeeping gene (HKG) [21]
While the use of an endogenous control is required for the correction of nonbiological and experimental variation, a non-optimal endogenous control can either introduce pseudo-variation or mask the real biological variation leading to misinterpretation of data

Summary

Introduction

Human epicardial adipose tissue (EAT) is a visceral fat depot that has gained significant attention in the recent times. A number of recent studies have, investigated the association between EAT expression of various adipokines, cytokines, oxidative stress- and inflammatory- markers with CAD [7,8,9,10]. For these studies, relative quantification of gene expression remains the method of choice. Our benign understanding of human EAT function would largely depend upon future studies assessing gene expression in this fat depot. 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

Objectives

Methods

Results

Conclusion