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

Abstract

<jats:p>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 (<jats:italic>Actb, Gapdh, Polr2a, Ywhag, Rpl13a, Sdha, Ppia, Tbp, Hprt1</jats:italic>and<jats:italic>Tfrc</jats:italic>) 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<jats:italic>Sdha, Tbp</jats:italic>, and<jats:italic>Hprt1</jats:italic>; in kidney,<jats:italic>Tbp</jats:italic><jats:italic>,</jats:italic><jats:italic>Actb</jats:italic>, and<jats:italic>Gapdh</jats:italic>were chosen, while<jats:italic>Actb</jats:italic><jats:italic>,</jats:italic><jats:italic>Ywhag</jats:italic>, and<jats:italic>Sdha</jats:italic>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.</jats:p>

Identification of Stable Reference Genes for Gene Expression Analysis of Three-Dimensional Cultivated Human Bone Marrow-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

“…39 In contrast, Cabiati et al identified TFRC as the least stable RG among ten candidates in cardiac and pulmonary tissues of obese and hyperglycemic rats. 40 The contrary results of TFRC gene expression stability depending on which species and tissue was analyzed show the importance of verifying the stability of internal control genes for each experimental setup. We do not recommend using TFRC as a RG for the combined analysis of 2D and 3D cultivated BM-MSCs, as the gene expression was significantly upregulated for BM-MSCs cultivated under 2D conditions.…”

Transcriptional Alterations of ET-1 Axis and DNA Damage in Lung Tissue of a Rat Obesity Model

“…Total RNA was extracted by the acid guanidinium thiocyanate-phenol-chloroform method using an RNeasy Midi kit (Qiagen) following the manufacturer's instructions, as previously described (Cabiati et al, 2012(Cabiati et al, , 2013. The RNA concentration and purity were determined spectrophotometrically (BioPhotometer, Eppendorf, Milano, Italy) measuring spectral absorption at 260 nm.…”

“…Real-Time polymerase chain reaction (PCR) reactions were performed in duplicate in the Bio-Rad C1000Ô thermal cycler (CFX-96 Real-Time PCR detection systems; Bio-Rad Laboratories), as previously described (Cabiati et al, 2012(Cabiati et al, , 2013. For monitoring cDNA amplification, a third-generation fluorophore, EvaGreen, was used (SsoFAST EvaGreen Supermix; Bio-Rad Laboratories).…”

“…Reaction conditions of all primer pairs used were optimized; that is, a gradient PCR was conducted to assess the optimal annealing temperature, while a standard curve obtained by scalar dilution of a cDNA pool (1:5, 1:25, 1:125, 1:625) was always generated to verify the PCR efficiency. The geometric mean of the three most stably expressed genes in the lung (ACTB, SDHA, YWHAG), established in a previous study (Cabiati et al, 2012), was used for normalization of Real-Time PCR results. Relative quantification of each target gene studied was calculated by the DDCt method using Bio-Rad's CFX96 manager software.…”

C-type atriuretic peptide causes relaxation of the internal anal sphincter through natriuretic peptide receptor B

“…Three housekeeping genes, GAPDH, HPRT1, and bactin, were used as endogenous controls to standardize the amount of cDNA. Thermal cycling conditions were one cycle of 2 minutes at 50 C and one cycle of 10 minutes at 95 C, followed by 40 cycles of amplification for 15 s at 95 C and 1 minute at 60 C. Cycle threshold values were obtained and quantification was performed by the relative expression method using the geometric mean of the housekeeping genes [27]. Negative controls had no reverse transcriptase added.…”