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Selection of reference genes for quantitative real‐time polymerase chain reaction studies in rat osteoblasts
Author(s) -
Abuna Rodrigo P. F.,
Oliveira Fabiola S.,
Ramos Jaqueline I. R.,
Lopes Helena B.,
Freitas Gileade P.,
Souza Alann T. P.,
Beloti Marcio M.,
Rosa Adalberto L.
Publication year - 2019
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.26886
Subject(s) - reference genes , runx2 , housekeeping gene , gene , bone marrow , osteoblast , gene expression , real time polymerase chain reaction , biology , mesenchymal stem cell , phenotype , polymerase chain reaction , microbiology and biotechnology , genetics , in vitro , immunology
Quantitative real‐time polymerase chain reaction (qRT‐PCR) is a powerful tool to evaluate gene expression, but its accuracy depends on the choice and stability of the reference genes used for normalization. In this study, we aimed to identify reference genes for studies on osteoblasts derived from rat bone marrow mesenchymal stem cells (bone marrow osteoblasts), osteoblasts derived from newborn rat calvarial (calvarial osteoblasts), and rat osteosarcoma cell line UMR‐106. The osteoblast phenotype was characterized by ALP activity and extracellular matrix mineralization. Thirty‐one candidates for reference genes from a Taqman ® array were assessed by qRT‐PCR, and their expressions were analyzed by five different approaches. The data showed that several of the most traditional reference genes, such as Actb and Gapdh , were inadequate for normalization and that the experimental conditions may affect gene stability. Eif2b1 was frequently identified among the best reference genes in bone marrow osteoblasts, calvarial osteoblasts, and UMR‐106 osteoblasts. Selected stable and unstable reference genes were used to normalize the gene expression of Runx2 , Alp , and Oc . The data showed statistically significant differences in the expression of these genes depending on the stability of the reference gene used for normalization, creating a bias that may induce incorrect assumptions in terms of osteoblast characterization of these cells. In conclusion, our study indicates that a rigorous selection of reference genes is a key step in qRT‐PCR studies in osteoblasts to generate precise and reliable data.