Abstract
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.
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Abbreviations
- Yrs:
-
years
- EAT:
-
epicardial adipose tissue
- B2M:
-
β2-microglobulin
- GAPDH:
-
Glyceraldehyde-3-phosphate dehydrogenase
- GUSB:
-
β-glucuronidase
- ACTB:
-
β-actin
- HPRT1:
-
Hypoxanthine phosphoribosyltransferase 1
- LRP10:
-
Low-density lipoprotein receptor-related protein
- PGK1:
-
Phosphoglycerate kinase 1
- RPII:
-
RNA polymerase II
- RPL13A:
-
Ribosomal protein L13A
- TFRC:
-
Transferrin receptor
- LV:
-
left ventricular myocardium
References
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. https://doi.org/10.1158/0008-5472.CAN-04-0496
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. https://doi.org/10.1371/journal.pone.0037517
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. https://doi.org/10.1186/1475-2840-5-1
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. https://doi.org/10.1210/jc.2007-2579
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. https://doi.org/10.1016/j.carpath.2010.07.005
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. https://doi.org/10.1007/s00380-012-0315-9
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. https://doi.org/10.1016/j.cjca.2012.06.003
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. https://doi.org/10.1016/j.cyto.2013.04.021
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. https://doi.org/10.1371/journal.pone.0032265
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. https://doi.org/10.1016/j.ijcard.2012.06.008
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. https://doi.org/10.1007/s11596-010-0547-9
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. https://doi.org/10.1016/j.cyto.2008.05.006
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. https://doi.org/10.1016/j.cyto.2010.05.009
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. https://doi.org/10.1038/sj.ijo.0803790
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. https://doi.org/10.1016/j.metabol.2009.12.027
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. https://doi.org/10.1186/1475-2840-10-87
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. https://doi.org/10.2169/internalmedicine.50.4753
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. https://doi.org/10.1536/ihj.52.139
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. https://doi.org/10.1016/j.jacc.2011.01.048
Huggett J et al (2005) Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6(4):279–284. https://doi.org/10.1038/sj.gene.6364190
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. https://doi.org/10.1038/oby.2009.374
Iacobellis G, Bianco AC (2011) Epicardial adipose tissue:emerging physiological, pathophysiological and clinical features. Trends Endocrinol Metab 22(11):450–457. https://doi.org/10.1016/j.tem.2011.07.003
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. https://doi.org/10.1016/S1885-5857(07)60081-4
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. https://doi.org/10.1016/j.bbrc.2013.01.078
Jaffer I, Riederer M, Shah P et al (2011) Expression of fat mobilizing genes in human epicardial adipose tissue. Atherosclerosis 220(1):122–127. https://doi.org/10.1016/j.atherosclerosis.2011.10.026
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):757–767
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. https://doi.org/10.1210/jc.2006-1044
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. https://doi.org/10.1152/ajpheart.00617.2009
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. https://doi.org/10.1139/Y11-014
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. https://doi.org/10.1152/ajpendo.90511.2008
Mazurek T, Zhang L, Zalewski A et al (2003) Human epicardial adipose tissue is a source of inflammatory mediators. Circulation 108(20):2460–2466. https://doi.org/10.1161/01.CIR.0000099542.57313.C5
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. https://doi.org/10.1186/1471-2199-11-39
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. https://doi.org/10.1007/s00592-012-0436-8
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. https://doi.org/10.1038/oby.2010.257
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. https://doi.org/10.1023/B:BILE.0000019559.84305.47
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. https://doi.org/10.1016/j.bbrc.2003.11.177
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):911–917
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. https://doi.org/10.1210/jc.2009-0571
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. https://doi.org/10.2337/dc10-2083
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. https://doi.org/10.1152/ajpheart.00120.2010
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. https://doi.org/10.1111/j.1365-2265.2011.04140.x
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. https://doi.org/10.1006/mcpr.2001.0376
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. https://doi.org/10.1016/j.regpep.2010.07.169
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. https://doi.org/10.1152/ajpendo.00273.2007
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. https://doi.org/10.5935/1678-9741.20110018
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. https://doi.org/10.1371/journal.pone.0041659
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. https://doi.org/10.1038/jhh.2008.75
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. https://doi.org/10.1016/j.cyto.2011.01.016
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. https://doi.org/10.1016/j.cyto.2012.08.012
Thellin O, Zorzi W, Lakaye B et al (1999) Housekeeping genes as internal standards: use and limits. J Biotechnol 75(2–3):291–295. https://doi.org/10.1016/S0168-1656(99)00163-7
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; https://books.google.sk/books?hl=en&lr=&id=YxGKpOg8TuQC&oi=fnd&pg=PA47&dq=Vandesompele+J,+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=false
Vokurka M, Lacinová Z, Kremen J et al (2010) Hepcidin expression in adipose tissue increase during cardiac surgery. Physiol Res 59(3):393–400
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. https://doi.org/10.1016/j.carpath.2008.02.006
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. https://doi.org/10.1186/1475-2840-10-2
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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.
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Mlynarova, J., Gazova, A., Musil, P. et al. Validation of reference genes in human epicardial adipose tissue and left ventricular myocardium in heart failure. Biologia 74, 1687–1698 (2019). https://doi.org/10.2478/s11756-019-00303-1
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DOI: https://doi.org/10.2478/s11756-019-00303-1