title: Tissue-specific selection of stable reference genes for real-time PCR normalization in an obese rat model.
abstract: 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 α, 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.
authors.affiliation: Laboratory of Cardiovascular Biochemistry, CNR Institute of Clinical Physiology, Via Giuseppe Moruzzi 1, 56124 Pisa, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Cabiati
authors.forename: Serena
authors.initials: S
authors.lastname: Raucci
authors.forename: Chiara
authors.initials: C
authors.lastname: Caselli
authors.forename: Maria Angela
authors.initials: MA
authors.lastname: Guzzardi
authors.forename: Andrea
authors.initials: A
authors.lastname: D'Amico
authors.forename: Tommaso
authors.initials: T
authors.lastname: Prescimone
authors.forename: Daniela
authors.initials: D
authors.lastname: Giannessi
authors.forename: Silvia
authors.initials: S
authors.lastname: Del Ry
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D020869:Gene Expression Profiling / Q000592:standards*; D005796:Genes; D006943:Hyperglycemia / Q000235:genetics; D008297:Male; D009765:Obesity / Q000235:genetics*; D009928:Organ Specificity / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha