, Volume 74, Issue 12, pp 1687–1698 | Cite as

Validation of reference genes in human epicardial adipose tissue and left ventricular myocardium in heart failure

  • Jana Mlynarova
  • Andrea GazovaEmail author
  • Peter Musil
  • Andrea Raganová
  • Ondrej Sprusansky
  • Eva Goncalvesova
  • Michal Hulman
  • John J. Leddy
  • Jan Kyselovic
Original Article


The characterization of tissue-specific gene expression in failing hearts requires the identification of appropriately validated reference genes. This study sought to validate which commonly used reference genes were the most suitable for qRT-PCR data normalization in epicardial adipose tissue and left ventricular myocardium in patients with end-stage heart failure. The mRNA expression of 10 candidate reference genes was analyzed by qRT-PCR. The stability of their expression was evaluated by our novel scoring system and compared with known algorithms used to identify stable reference genes (Normfinder and Bestkeeper). Using these three methods, we selected the Ribosomal protein L13A (RPL13A) as the best reference gene when studying either epicardial adipose tissue or left ventricular myocardium in failing hearts. In contrast, the β2-microglobulin (B2M) gene was identified as a more suitable reference gene in studies comparing gene expression in epicardial adipose tissue to left ventricular myocardium.


qRT-PCR normalization Ribosomal protein L13A β2-microglobulin 





epicardial adipose tissue




Glyceraldehyde-3-phosphate dehydrogenase






Hypoxanthine phosphoribosyltransferase 1


Low-density lipoprotein receptor-related protein


Phosphoglycerate kinase 1


RNA polymerase II


Ribosomal protein L13A


Transferrin receptor


left ventricular myocardium



This publication is the result of the project implementation: Biomakro ITMS: 26240120027, VEGA 1/0905/14, VEGA 1/0949/15, APVV-14-0416.

Special thanks to colleagues Peter Krenek, Gabriel Dóka and Lenka Pivackova.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.


  1. Andersen CL, Jensen JL, Ørntoft TF (2004) Normalization of real-time Qunatitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and Colon Cancer data sets. Cancer Res 64(15):5245–5250. CrossRefPubMedGoogle Scholar
  2. Arsenijevic T et al (2012) Murine 3T3-L1 adipocyte cell differentiation model: validation reference genes for qPCR gene expression analysis. PLoS One 7(5):e37517. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Baker AR, Silva NF, Quinn DW et al (2006) Human epicardial adipose tissue expresses a pathogenic profile of adipocytokines in patients with cardiovascular disease. Cardiovasc Diabetol 5:1. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Baker AR, Harte AL, Howell N et al (2009) Epicardial adipose tissue as a source of nuclear factor-{kappa} B and c-Jun N-terminal kinase mediated inflammation in patients with coronary artery disease. J Clin Endocrinol Metab 94(1):261–267. CrossRefPubMedGoogle Scholar
  5. Bambace C, Telesca M, Zoico E et al (2011) Adiponectin gene expression and adipocyte diameter: a comparison between epicardial and subcutaneous adipose tissue in men. Cardiovasc Pathol 20(5):e153–e156. CrossRefPubMedGoogle Scholar
  6. Bambace C, Sepe A, Zoico E et al (2013) Inflammatory profile in subcutaneous and epicardial adipose tissue in men with and without diabetes. Heart Vessel 29(1):42–48. CrossRefGoogle Scholar
  7. Cappellano G, Uberti F, Caimmi PP et al (2013) Different expression and function of the endocannabinoid system in human epicardial adipose tissue in relation to heart disease. Can J Cardiol 29(4):499–509. CrossRefPubMedGoogle Scholar
  8. Caselli C, D'Amico A, Caruso R et al (2013) Impact of normalization strategy on cardiac expression of pro-inflammatory cytokines: evaluation of reference genes in different human myocardial regions after left ventricular assist device support. Cytokine 63(2):113–122. CrossRefPubMedGoogle Scholar
  9. Chechi K et al (2012) Validation of reference genes for the relative quantification of gene expression in human Epicardial adipose tissue. PLoS One 7(4):e32265. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Chechi K, Blanchard PG, Mathieu P, Deshaies Y, Richard D (2013) Brown fat like gene expression in the epicardial fat depot correlates with circulating HDL-cholesterol and triglycerides in patients with coronary artery disease. Int J Cardiol 167(5):2264–2270. CrossRefPubMedGoogle Scholar
  11. Chen X, Jiao Z, Wang L et al (2010) Roles of human epicardial adipose tissue in coronary artery atherosclerosis. J Huazhong Univ Sci Technolog Med Sci 30(5):589–593. CrossRefPubMedGoogle Scholar
  12. Eiras S, Teijeira-Fernández E, Shamagian LG et al (2008) Extension of coronary artery disease is associated with increased IL-6 and decreased adiponectin gene expression in epicardial adipose tissue. Cytokine 43(2):174–180. CrossRefPubMedGoogle Scholar
  13. Eiras S, Teijeira-Fernández E, Salgado-Somoza A et al (2010) Relationship between epicardial adipose tissue adipocyte size and MCP-1 expression. Cytokine 51(2):207–212. CrossRefPubMedGoogle Scholar
  14. Fain JN, Sacks HS, Buehrer B et al (2008) Identification of omentin mRNA in human epicardial adipose tissue: comparison to omentin in subcutaneous, internal mammary artery periadventitial and visceral abdominal depots. Int J Obes 32(5):810–815. CrossRefGoogle Scholar
  15. Fain JN, Sacks HS, Bahouth SW et al (2010) Human epicardial adipokine messenger RNAs: comparisons of their expression in substernal, subcutaneous, and omental fat. Metabolism 59(9):1379–1386. CrossRefPubMedGoogle Scholar
  16. Gao X, Mi S, Zhang F et al (2011) Association of chemerin mRNA expression in human epicardial adipose tissue with coronary atherosclerosis. Cardiovasc Diabetol 10:87. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Gormez S, Demirkan A, Atalar F et al (2011) Adipose tissue gene expression of adiponectin, tumor necrosis factor-α and leptin in metabolic syndrome patients with coronary artery disease. Intern Med 50(8):805–810. CrossRefPubMedGoogle Scholar
  18. Hirata Y, Kurobe H, Akaike M et al (2011a) Enhanced inflammation in epicardial fat in patients with coronary artery disease. Int Heart J 52(3):139–142. CrossRefPubMedGoogle Scholar
  19. Hirata Y, Tabata M, Kurobe H et al (2011b) Coronary atherosclerosis is associated with macrophage polarization in epicardial adipose tissue. J Am Coll Cardiol 58(3):248–255. CrossRefPubMedGoogle Scholar
  20. Huggett J et al (2005) Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6(4):279–284. CrossRefPubMedGoogle Scholar
  21. Hurtado del Pozo C, Calvo RM, Vesperinas-García G et al (2010) IPO8 and FBXL10: new reference genes for gene expression studies in human adipose tissue. Obesity 18(5):897–903. CrossRefPubMedGoogle Scholar
  22. Iacobellis G, Bianco AC (2011) Epicardial adipose tissue:emerging physiological, pathophysiological and clinical features. Trends Endocrinol Metab 22(11):450–457. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Iglesias MJ, Eiras S, Piñeiro R et al (2006) Gender differences in adiponectin and leptin expression in epicardial and subcutaneous adipose tissue. Findings in patients undergoing cardiac surgery. Rev Esp Cardiol 59(12):1252–1260. CrossRefPubMedGoogle Scholar
  24. Imoto-Tsubakimoto H, Takahashi T, Ueyama T et al (2013) Serglycin is a novel adipocytokine highly expressed in epicardial adipose tissue. Biochem Biophys Res Commun 432:105–110. CrossRefPubMedGoogle Scholar
  25. Jaffer I, Riederer M, Shah P et al (2011) Expression of fat mobilizing genes in human epicardial adipose tissue. Atherosclerosis 220(1):122–127. CrossRefPubMedGoogle Scholar
  26. Kotulák T, Drápalová J, Kopecký P et al (2011) Increased circulating and epicardial adipose tissue mRNA expression of fibroblast growth factor - 21 after cardiac surgery: a possible role in postoperative inflammatory response and insulin resistance. Physiol Res 60(5):757–767PubMedGoogle Scholar
  27. Kremen J, Dolinkova M, Krajickova J et al (2006) Increased subcutaneous and epicardial adipose tissue production of proinflammatory cytokines in cardiac surgery patients: possible role in postoperative insulin resistance. J Clin Endocrinol Metab 91(11):4620–4627. CrossRefPubMedGoogle Scholar
  28. Langheim S, Dreas L, Veschini L et al (2010) Increased expression and secretion of resistin in epicardial adipose tissue of patients with acute coronary syndrome. Am J Physiol Heart Circ Physiol 298(3):H746–H753. CrossRefPubMedGoogle Scholar
  29. Mackovicova K, Gazova A, Kucerova D et al (2011) Enalapril decreases cardiac mass and fetal gene expression without affecting the expression of endothelin-1, transforming growth factor beta-1, or cardiotrophin-1 in the healthy normotensive rat. Can. J Physiol Pharmacol 89(3):197–205. CrossRefGoogle Scholar
  30. Madani R, Karastergiou K, Ogston NC et al (2009) RANTES release by human adipose tissue in vivo and evidence for depot-specific differences. Am J Physiol Endocrinol Metab 296(6):E1262–E1268. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Mazurek T, Zhang L, Zalewski A et al (2003) Human epicardial adipose tissue is a source of inflammatory mediators. Circulation 108(20):2460–2466. CrossRefPubMedGoogle Scholar
  32. Mehta R, Birerdinc A, Hossain N et al (2010) Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples. BMC Mol Biol 11:39. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Nasarre L, Juan-Babot O, Gastelurrutia P et al (2012) Low density lipoprotein receptors-related protein 1 is upregulated in epicardial fat from type 2 diabetes mellitus patients and correlates with glucose and triglyceride plasma levels. Acta Diabetol 51(1):23–30. CrossRefPubMedGoogle Scholar
  34. Neville MJ, Collins JM, Gloyn AL et al (2011) Comprehensive human adipose tissue mRNA and MicroRNA endogenous control selection for quantitative real-time PCR normalization. Obesity 19(4):888–892. CrossRefPubMedGoogle Scholar
  35. Pfaffl MW et al (2004) Determination of stable housekeeping genes, differentially regulated target genes and samples integrity: BestKeeper- excel-based tool using pair-wise correlations. Biotechnol Lett 26(6):509–515. CrossRefPubMedGoogle Scholar
  36. Radonić A, Thulke S, Mackay IM et al (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 313(14):856–862. CrossRefPubMedGoogle Scholar
  37. Roubícek T, Dolinková M, Bláha J et al (2008) Increased angiotensinogen production in epicardial adipose tissue during cardiac surgery: possible role in a postoperative insulin resistance. Physiol Res 57(6):911–917PubMedGoogle Scholar
  38. Sacks HS, Fain JN, Holman B et al (2009) Uncoupling protein-1 and related messenger ribonucleic acids in human epicardial and other adipose tissues: Epicardial fat functioning as brown fat. J Clin Endocrinol Metab 94(9):3611–3615. CrossRefPubMedGoogle Scholar
  39. Sacks HS, Fain JN, Cheema P et al (2011) Inflammatory genes in epicardial fat contiguous with coronary atherosclerosis in the metabolic syndrome and type 2 diabetes. Diabetes Care 34(3):730–733. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Salgado-Somoza A, Teijeira-Fernández E, Fernández AL, González-Juanatey JR, Eiras S (2010) Proteomic analysis of epicardial and subcutaneous adipose tissue reveals differences in proteins involved on oxidative stress. Am J Physiol Heart Circ Physiol 299(1):H202–H209. CrossRefPubMedGoogle Scholar
  41. Salgado-Somoza A, Teijeira-Fernández E, Rubio J et al (2012) Coronary artery disease is associated with higher epicardial retinol-binding protein 4 (RBP4) and lower glucose transporter (GLUT) 4 levels in epicardial and subcutaneous adipose tissue. Clin Endocrinol 76(1):51–58. CrossRefGoogle Scholar
  42. Selvey S, Thompson EW, Matthaei K et al (2001) β-Actin- an unsuitable internal control for RT-PCR. Mol Cell Probes 15(5):307–311. CrossRefPubMedGoogle Scholar
  43. Shibasaki I, Nishikimi T, Mochizuki Y et al (2010) Greater expression of inflammatory cytokine, adrenomedullin, and natriuretic peptide receptor-C in epicardial adipose tissue in coronary artery disease. Regul Pept 165(2–3):210–217. CrossRefPubMedGoogle Scholar
  44. Silaghi A, Achard V, Paulmyer-Lacroix O et al (2007) Expression of adrenomedullin in human epicardial adipose tissue: role of coronary status. Am J Physiol Endocrinol Metab 293(5):E1443–E1450. CrossRefPubMedGoogle Scholar
  45. Spener RF, Breda JR, Pires AC, Pinhal MA, Souto RP (2011) Adiponectin expression in epicardial adipose tissue after percutaneous coronary intervention with bare-metal stent. Rev Bras Cir Cardiovasc 26(3):427–432. CrossRefPubMedGoogle Scholar
  46. Sun Y, Li Y, Luo D, Liao DJ (2012) Pseudogenes as weaknesses of ACTB (Actb) and GAPDH (Gapdh) used as reference genes in reverse transcription and polymerase chain reactions. PLoS One 7(8):e41659. CrossRefPubMedPubMedCentralGoogle Scholar
  47. Teijeira-Fernandez E, Eiras S, Grigorian-Shamagian L, Fernandez A, Adrio B, Gonzalez-Juanatey JR (2008) Epicardial adipose tissue expression of adiponectin is lower in patients with hypertension. J Hum Hypertens 22(12):856–863. CrossRefPubMedGoogle Scholar
  48. Teijeira-Fernandez E, Eiras S, Shamagian LG et al (2011) Lower epicardial adipose tissue adiponectin in patients with metabolic syndrome. Cytokine 54(2):185–190. CrossRefPubMedGoogle Scholar
  49. Teijeira-Fernandez E, Eiras S, Salgado Somoza A, Gonzalez-Juanatey JR (2012) Baseline epicardial adipose tissue levels predict cardiovascular outcomes: a long-term follow-up study. Cytokine 60(3):674–680. CrossRefPubMedGoogle Scholar
  50. Thellin O, Zorzi W, Lakaye B et al (1999) Housekeeping genes as internal standards: use and limits. J Biotechnol 75(2–3):291–295. CrossRefPubMedGoogle Scholar
  51. Vandesompele J, Kubista M, Pfaffl MW (2009) Reference Gene Validation Software for Improved Normalization. In: Logan J, Edwards K, Saunders N (ed) Real-Time PCR: Current Technology and Applications, 1st edn. Caister Academic Press, Norfolk, pp. 47–64;,+Kubista+M,+Pfaffl+MW:+Reference+gene+validation+software+for+improved+normalization.+Real-time+PCR:+An+Essential+Guide.+Edited+by:+Edwards+K,+Logan+J,+Saunders+N.+Horizon+Scientific+Press%3B+Norwich,+2,&ots=OQjkm4hRSo&sig=e5F2Oz1_dEGY7hOzG70O9XqkuAM&redir_esc=y#v=onepage&q&f=falseGoogle Scholar
  52. Vokurka M, Lacinová Z, Kremen J et al (2010) Hepcidin expression in adipose tissue increase during cardiac surgery. Physiol Res 59(3):393–400PubMedGoogle Scholar
  53. Vural B, Atalar F, Ciftci C et al (2008) Presence of fatty-acid-binding protein 4 expression in human epicardial adipose tissue in metabolic syndrome. Cardiovasc Pathol 17(6):392–398. CrossRefPubMedGoogle Scholar
  54. Zhou Y, Wei Y, Wang L et al (2011) Decreased adiponectin and increased inflammation expression in epicardial adipose tissue in coronary artery disease. Cardiovasc Diabetol 10(1):2. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Institute of Molecular Biology, Slovak Academy of Sciences 2019 2019

Authors and Affiliations

  1. 1.V. Department of Internal MedicineFaculty of Medicine, Comenius UniversityBratislavaSlovakia
  2. 2.Institute of Pharmacology and Clinical PharmacologyFaculty of Medicine, Comenius UniversityBratislavaSlovakia
  3. 3.Department of Pharmacology and Toxicology, Faculty of PharmacyComenius UniversityBratislavaSlovakia
  4. 4.National Institute for Cardiovascular DiseasesBratislavaSlovakia
  5. 5.Department of Cellular and Molecular Medicine, Faculty of MedicineUniversity of OttawaOttawaCanada

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