title: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Centre de Recherche de l'Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Québec, Canada.
authors.forename: Kanta
authors.initials: K
authors.lastname: Chechi
authors.forename: Yves
authors.initials: Y
authors.lastname: Gelinas
authors.forename: Patrick
authors.initials: P
authors.lastname: Mathieu
authors.forename: Yves
authors.initials: Y
authors.lastname: Deshaies
authors.forename: Denis
authors.initials: D
authors.lastname: Richard
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000273:Adipose Tissue / Q000378:metabolism*; D000465:Algorithms; D015331:Cohort Studies; D020869:Gene Expression Profiling; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism; D010496:Pericardium / Q000378:metabolism*; D012318:RNA Polymerase I / Q000235:genetics / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012016:Reference Values; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D012984:Software; D001613:beta 2-Microglobulin / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; D012337:RNA, Ribosomal, 18S; D012269:Ribosomal Proteins; D001613:beta 2-Microglobulin; C054675:ribosomal protein P0; C034661:ribosomal proteins L27; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); D007041:Hypoxanthine Phosphoribosyltransferase; D012318:RNA Polymerase I; D019696:Peptidylprolyl Isomerase