title: Identification of valid reference genes for the normalization of RT-qPCR expression studies in human breast cancer cell lines treated with and without transient transfection.
abstract: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique for examining gene expression changes during tumorigenesis. Target gene expression is generally normalized by a stably expressed endogenous reference gene; however, reference gene expression may differ among tissues under various circumstances. Because no valid reference genes have been documented for human breast cancer cell lines containing different cancer subtypes treated with transient transfection, we identified appropriate and reliable reference genes from thirteen candidates in a panel of 10 normal and cancerous human breast cell lines under experimental conditions with/without transfection treatments with two transfection reagents. Reference gene expression stability was calculated using four algorithms (geNorm, NormFinder, BestKeeper and comparative delta Ct), and the recommended comprehensive ranking was provided using geometric means of the ranking values using the RefFinder tool. GeNorm analysis revealed that two reference genes should be sufficient for all cases in this study. A stability analysis suggests that 18S rRNA-ACTB is the best reference gene combination across all cell lines; ACTB-GAPDH is best for basal breast cancer cell lines; and HSPCB-ACTB is best for ER+ breast cancer cells. After transfection, the stability ranking of the reference gene fluctuated, especially with Lipofectamine 2000 transfection reagent in two subtypes of basal and ER+ breast cell lines. Comparisons of relative target gene (HER2) expression revealed different expressional patterns depending on the reference genes used for normalization. We suggest that identifying the most stable and suitable reference genes is critical for studying specific cell lines under certain circumstances.
authors.affiliation: Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming, China.
authors.forename: Lin-Lin
authors.initials: LL
authors.lastname: Liu
authors.affiliation: Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming, China.
authors.forename: Teng-Fei
authors.initials: TF
authors.lastname: Ma
authors.affiliation: Department of Endocrine Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China; Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Ge
authors.affiliation: Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China.
authors.forename: Ce-Shi
authors.initials: CS
authors.lastname: Chen
authors.affiliation: Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming, China; State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China.
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Zhang
mesh_terms: D000465:Algorithms; D001943:Breast Neoplasms / Q000473:pathology*; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics; D020869:Gene Expression Profiling; D018734:Genes, erbB-2 / Q000235:genetics; D006801:Humans; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D014162:Transfection
chemical_list: D017931:DNA Primers