RESUMO
Accurate normalization is a primary component of a reliable gene expression analysis based on qRT-PCR technique. While the use of one or more reference genes as internal controls is commonly accepted as the most appropriate normalization strategy, many qPCR-based published studies still contain data poorly normalized and reference genes arbitrarily chosen irrespective of the particular tissue and the specific experimental design. To date, no validated reference genes have been identified for endometrial cancer tissues. In this study, 10 normalization genes (GAPDH, B2M, ACTB, POLR2A, UBC, PPIA, HPRT1, GUSB, TBP, H3F3A) belonging to different functional and abundance classes in various tissues and used in different studies, were analyzed to determine their applicability. In total, 100 endometrioid endometrial cancer samples, which were carefully balanced according to their tumor grade, and 29 normal endometrial tissues were examined using SYBR Green Real-Time RT-PCR. The expression stability of candidate reference genes was determined and compared by means of geNorm and NormFinder softwares. Both algorithms were in agreement in identifying GAPDH, H3F3A, PPIA, and HPRT1 as the most stably expressed genes, only differing in their ranking order. Analysis performed on the expression levels of all candidate genes confirm HPRT1 and PPIA as the most stably expressed in the study groups regardless of sample type, to be used alone or better in combination. As the stable expression of HPRT1 and PPIA between normal and tumor endometrial samples fulfill the basic requirement of a reference gene to be used for normalization purposes, HPRT1 expression showed significant differences between samples from low-grade and high-grade tumors. In conclusion, our results recommend the use of PPIA as a single reference gene to be considered for improved reliability of normalization in gene expression studies involving endometrial tumor samples at different tumor degrees.
Assuntos
Carcinoma Endometrioide/genética , Neoplasias Ovarianas/genética , Actinas/genética , Actinas/normas , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Endometrioide/metabolismo , Carcinoma Endometrioide/patologia , Estudos de Coortes , Endométrio/metabolismo , Endométrio/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/normas , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , RNA/isolamento & purificação , RNA/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , Valores de Referência , SoftwareRESUMO
Accurate quantification of gene expression with quantitative real-time polymerase chain reaction (qRT-PCR) relies on the choice of an appropriate reference gene. In this study, nine candidate reference genes were selected to study the expression stability for qRT-PCR normalization in adventitious rooting of Populus hardwood cuttings. geNorm, NormFinder, and BestKeeper analysis revealed that actin isoform B (ACT) was the most unstable gene across developmental stages, whereas elongation factor 1 alpha (EF1a) and 18S recombinant RNA (18S) emerged as the most appropriate reference genes for qRT-PCR analysis in this complex developmental process.
Assuntos
Populus/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Actinas/genética , Actinas/normas , Sequência de Bases , Genes de Plantas , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/normas , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/normas , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normasRESUMO
BACKGROUND: Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. RESULTS: Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes. CONCLUSIONS: We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.
