Impact of reference gene selection for target gene normalization on experimental outcome using real-time qRT-PCR in adipocytes.
Impact of reference gene selection for target gene normalization on experimental outcome using real-time qRT-PCR in adipocytes.
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DOI：
10.1371/journal.pone.0015208
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发表时间：
2010-12-13
期刊：
影响因子：
3.7
通讯作者：
Morrison RF
中科院分区：
文献类型：
--
作者：
Ferguson BS;Nam H;Hopkins RG;Morrison RF
随着当前肥胖相关疾病的增加，实时定量逆转录聚合酶链反应（qRT-PCR）已成为一种广泛使用的评估脂肪细胞表达和调控基因的方法。为了准确测量相对基因表达的变化并监测样本间的可变性，在qRT-PCR测定中通常使用对相对表达不变化的内源控制基因进行归一化。然而，历史证据清楚地表明，传统的控制基因（如β-肌动蛋白、GAPDH、α-微管蛋白）的表达谱在不同的组织类型和实验条件下受到不同的调节。因此，我们利用TaqMan qRT-PCR验证了3T3-L1脂肪细胞在炎症应激、氧化应激、细胞周期同步进展和细胞分化等不同实验条件下常用的6种内源性调控基因。在每种研究条件下，我们通过与脂肪细胞功能和分化相关的靶基因的例子进一步评估内参基因选择对实验结果的影响。我们证明了多个内参基因以一种条件特异性的方式进行调节，这种方式不适合用于靶基因规范化。数据表明，不适当的参考基因选择可以对研究结论产生深远的影响，从不同的统计结果到显著程度的不准确数据解释。本研究验证了在3T3-L1脂肪细胞中使用内源性对照，并强调了不适当的内参基因选择对数据解释和研究结论的影响。
With the current rise in obesity-related morbidities, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become a widely used method for assessment of genes expressed and regulated by adipocytes. In order to measure accurate changes in relative gene expression and monitor intersample variability, normalization to endogenous control genes that do not change in relative expression is commonly used with qRT-PCR determinations. However, historical evidence has clearly demonstrated that the expression profiles of traditional control genes (e.g., β-actin, GAPDH, α-tubulin) are differentially regulated across multiple tissue types and experimental conditions. Therefore, we validated six commonly used endogenous control genes under diverse experimental conditions of inflammatory stress, oxidative stress, synchronous cell cycle progression and cellular differentiation in 3T3-L1 adipocytes using TaqMan qRT-PCR. Under each study condition, we further evaluated the impact of reference gene selection on experimental outcome using examples of target genes relevant to adipocyte function and differentiation. We demonstrate that multiple reference genes are regulated in a condition-specific manner that is not suitable for use in target gene normalization. Data are presented demonstrating that inappropriate reference gene selection can have profound influence on study conclusions ranging from divergent statistical outcome to inaccurate data interpretation of significant magnitude. This study validated the use of endogenous controls in 3T3-L1 adipocytes and highlights the impact of inappropriate reference gene selection on data interpretation and study conclusions.
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