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首页> 外文期刊>Naunyn-Schmiedeberg's Archives of Pharmacology >Rifampicin alters the expression of reference genes used to normalize real-time quantitative RT-PCR data
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Rifampicin alters the expression of reference genes used to normalize real-time quantitative RT-PCR data

机译：利福平改变用于标准化实时定量RT-PCR数据的参考基因的表达

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摘要

Suitable reference genes for correct quantification of reverse transcription PCR (qRT-PCR) have to be constantly expressed in all samples under investigation. Thus, it is mandatory to determine expression stability of control genes before normalization. We aimed to establish optimum inducing concentrations for the prototypical enzyme and drug transporter inducer rifampicin in LS180 cells and concurrently assessed reference gene stability under rifampicin treatment. LS180 cells were treated with increasing concentrations of rifampicin (up to 200 μM), and expression of eight different reference genes and some target genes (CYP3A4, ABCB1, and ABCC1) was quantified using real-time qRT-PCR. To check whether the results can be generalized, HepG2 cells were also investigated. We demonstrated that higher concentrations of rifampicin (50 μM) change the expression of reference genes and thus may complicate and adulterate normalization of qRT-PCR data. The results stress the need for proper validation of potential reference genes in respective cells, tissues, and particular experimental conditions. Programs like geNorm and NormFinder alone do not warrant an adequate choice of the most suitable reference gene. Scrutiny of the reference gene expression and plausibility of the data remain necessary and protect from erroneous quantification and misinterpretation of qRT-PCR data.
机译：用于反转录PCR（qRT-PCR）正确定量的合适参考基因必须在所有正在研究的样品中不断表达。因此，必须在标准化之前确定对照基因的表达稳定性。我们的目的是为LS180细胞中原型酶和药物转运诱导剂利福平建立最佳诱导浓度，并同时评估利福平处理下的参考基因稳定性。 LS180细胞用递增浓度的利福平（最高200μM）处理，并使用实时qRT-PCR定量了八个不同参考基因和一些靶基因（CYP3A4，ABCB1和ABCC1）的表达。为了检查结果是否可以概括，还研究了HepG2细胞。我们证明，较高浓度的利福平（> 50μM）会改变参考基因的表达，因此可能会使qRT-PCR数据的标准化复杂化和掺假。结果强调需要适当验证各个细胞，组织和特定实验条件下的潜在参考基因。单独使用诸如geNorm和NormFinder这样的程序并不能保证对最合适的参考基因进行适当的选择。仔细检查参考基因的表达和数据的真实性仍然是必要的，可以防止qRT-PCR数据的错误定量和误解。

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  • 来源
    《Naunyn-Schmiedeberg's Archives of Pharmacology》 |2012年第10期|p.1025-1034|共10页
  • 作者

    Johanna Weiss; Dirk Theile; Walter Emil Haefeli;

  • 作者单位

    Department of Clinical Pharmacology and Pharmacoepidemiology, University Hospital of Heidelberg, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany;

    Department of Clinical Pharmacology and Pharmacoepidemiology, University Hospital of Heidelberg, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany;

    Department of Clinical Pharmacology and Pharmacoepidemiology, University Hospital of Heidelberg, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany;

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  • 原文格式 PDF
  • 正文语种 eng
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  • 关键词

    Rifampicin; Induction; Reference gene; PCR; CYP;

    机译：利福平;诱导;参考基因;PCR;CYP;

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