Tissue-specific selection of stable reference genes for real-time PCR normalization in an obese rat model

被引：50

作者：

Cabiati, Manuela ^{[1
]}

Raucci, Serena ^{[1
]}

Caselli, Chiara ^{[1
]}

Guzzardi, Maria Angela ^{[1
]}

D'Amico, Andrea ^{[1
]}

Prescimone, Tommaso ^{[1
]}

Giannessi, Daniela ^{[1
]}

Del Ry, Silvia ^{[1
]}

机构：

[1] CNR Inst Clin Physiol, Lab Cardiovasc Biochem, I-56124 Pisa, Italy

来源：

JOURNAL OF MOLECULAR ENDOCRINOLOGY | 2012年 / 48卷 / 03期

关键词：

HEART-FAILURE; RT-PCR; EXPRESSION; ALPHA; HYPERTENSION; DYSFUNCTION;

D O I：

10.1530/JME-12-0024

中图分类号：

R5 [内科学];

学科分类号：

1002 ; 100201 ;

摘要：

Obesity is a complex pathology with interacting and confounding causes due to the environment, hormonal signaling patterns, and genetic predisposition. At present, the Zucker rat is an eligible genetic model for research on obesity and metabolic syndrome, allowing scrutiny of gene expression profiles. Real-time PCR is the benchmark method for measuring mRNA expressions, but the accuracy and reproducibility of its data greatly depend on appropriate normalization strategies. In the Zucker rat model, no specific reference genes have been identified in myocardium, kidney, and lung, the main organs involved in this syndrome. The aim of this study was to select among ten candidates (Actb, Gapdh, Polr2a, Ywhag, Rpl13a, Sdha, Ppia, Tbp, Hprt1 and Tfrc) a set of reference genes that can be used for the normalization of mRNA expression data obtained by real-time PCR in obese and lean Zucker rats both at fasting and during acute hyperglycemia. The most stable genes in the heart were Sdha, Tbp, and Hprt1; in kidney, Tbp, Actb, and Gapdh were chosen, while Actb, Ywhag, and Sdha were selected as the most stably expressed set for pulmonary tissue. The normalization strategy was used to analyze mRNA expression of tumor necrosis factor a, the main inflammatory mediator in obesity, whose variations were more significant when normalized with the appropriately selected reference genes. The findings obtained in this study underline the importance of having three stably expressed reference gene sets for use in the cardiac, renal, and pulmonary tissues of an experimental model of obese and hyperglycemic Zucker rats. Journal of Molecular Endocrinology (2012) 48, 251-260

引用

页码：251 / 260

页数：10

共 37 条

[1]

Antic Vladan, 2003, Heart Lung Circ, V12, P84

[2] Expression of mRNA encoding G protein-coupled receptors involved in congestive heart failure - A quantitative RT-PCR study and the question of normalisation [J].

Brattelid, Trond ;

Tveit, Kristine ;

Birkeland, Jon Arne K. ;

Sjaastad, Ivar ;

Qvigstad, Eirik ;

Krobert, Kurt Allen ;

Hussain, Rizwan I. ;

Skomedal, Tor ;

Osnes, Jan-Bjorn ;

Levy, Finn Olav .

BASIC RESEARCH IN CARDIOLOGY, 2007, 102 (03) :198-208

[3]

BRAY GA, 1977, FED PROC, V36, P148

[4] Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays [J].

Bustin, SA .

JOURNAL OF MOLECULAR ENDOCRINOLOGY, 2000, 25 (02) :169-193

[5] The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments [J].

Bustin, Stephen A. ;

Benes, Vladimir ;

Garson, Jeremy A. ;

Hellemans, Jan ;

Huggett, Jim ;

Kubista, Mikael ;

Mueller, Reinhold ;

Nolan, Tania ;

Pfaffl, Michael W. ;

Shipley, Gregory L. ;

Vandesompele, Jo ;

Wittwer, Carl T. .

CLINICAL CHEMISTRY, 2009, 55 (04) :611-622

[6] Reference genes for normalising gene expression data in collagenase-induced rat intracerebral haemorrhage [J].

Cook, Naomi L. ;

Kleinig, Timothy J. ;

van den Heuvel, Corinna ;

Vink, Robert .

BMC MOLECULAR BIOLOGY, 2010, 11

[7]

Cozzi Jean, 2009, Methods Mol Biol, V561, P73, DOI 10.1007/978-1-60327-019-9_5

[8] Evidence Based Selection of Housekeeping Genes [J].

de Jonge, Hendrik J. M. ;

Fehrmann, Rudolf S. N. ;

de Bont, Eveline S. J. M. ;

Hofstra, Robert M. W. ;

Gerbens, Frans ;

Kamps, Willem A. ;

de Vries, Elisabeth G. E. ;

van der Zee, Ate G. J. ;

Meerman, Gerard J. te ;

ter Elst, Arja .

PLOS ONE, 2007, 2 (09)

[9] Differential expression of GAPDH and β-actin in growing collateral arteries [J].

Deindl, E ;

Boengler, K ;

van Royen, N ;

Schaper, W .

MOLECULAR AND CELLULAR BIOCHEMISTRY, 2002, 236 (1-2) :139-146

[10] TNF-α impairs insulin signaling and insulin stimulation of glucose uptake in C2C12 muscle cells [J].

Del Aguila, LF ;

Claffey, KP ;

Kirwan, JP .

AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM, 1999, 276 (05) :E849-E855

← 1 2 3 4 →