Validation of endogenous control reference genes for normalizing gene expression studies in endometrial carcinoma.
Mol Hum Reprod
; 21(9): 723-35, 2015 Sep.
Article
em En
| MEDLINE
| ID: mdl-26124453
ABSTRACT
Real-time quantitative RT-PCR (qRT-PCR) is a powerful technique used for the relative quantification of target genes, using reference (housekeeping) genes for normalization to ensure the generation of accurate and robust data. A systematic examination of the suitability of endogenous reference genes for gene expression studies in endometrial cancer tissues is absent. The aims of this study were therefore to identify and evaluate from the thirty-two possible reference genes from a TaqMan(®) array panel their suitability as an internal control gene. The mathematical software packages geNorm qBasePLUS identified Pumilio homolog 1 (Drosophila) (PUM1), ubiquitin C (UBC), phosphoglycerate kinase (PGK1), mitochondrial ribosomal protein L19 (MRPL19) and peptidylpropyl isomerase A (cyclophilin A) (PPIA) as the best reference gene combination, whilst NormFinder identified MRPL19 as the best single reference gene, with importin 8 (IPO8) and PPIA being the best combination of two reference genes. BestKeeper ranked MRPL19 as the most stably expressed gene. In addition, the study was validated by examining the relative expression of a test gene, which encodes the cannabinoid receptor 1 (CB1). A significant difference in CB1 mRNA expression between malignant and normal endometrium using MRPL19, PPIA, and IP08 in combination was observed. The use of MRPL19, IPO8 and PPIA was identified as the best reference gene combination for the normalization of gene expression levels in endometrial carcinoma. This study demonstrates that the arbitrary selection of endogenous control reference genes for normalization in qRT-PCR studies of endometrial carcinoma, without validation, risks the production of inaccurate data and should therefore be discouraged.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Biomarcadores Tumorais
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Neoplasias do Endométrio
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Carcinoma Endometrioide
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Perfilação da Expressão Gênica
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Reação em Cadeia da Polimerase em Tempo Real
Tipo de estudo:
Observational_studies
/
Prognostic_studies
/
Risk_factors_studies
Limite:
Female
/
Humans
Idioma:
En
Revista:
Mol Hum Reprod
Assunto da revista:
BIOLOGIA MOLECULAR
/
MEDICINA REPRODUTIVA
Ano de publicação:
2015
Tipo de documento:
Article
País de afiliação:
Reino Unido