Equivalence test in quantitative reverse transcription polymerase chain reaction:: confirmation of reference genes suitable for normalization
Equivalence test in quantitative reverse transcription polymerase chain reaction:: confirmation of reference genes suitable for normalization
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DOI：
10.1016/j.ab.2004.08.024
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发表时间：
2004-12-01
影响因子：
2.9
通讯作者：
Füzesi, L
中科院分区：
文献类型：
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作者：
Haller, F;Kulle, B;Füzesi, L
在定量逆转录-聚合酶链反应（qRT-PCR）中，使用参考基因的标准化是一种常用的有用方法，但合适的参考基因的验证仍然是一个关键问题。使用未经证实的参考基因可能导致对靶基因表达的误解。本研究的目的是采用适当的统计方法来鉴定和验证适用于qRT-PCR试验标准化的参考基因。我们介绍了稳定表达的参考基因鉴定的等效性测试。为了评价该试验的优点，在不同组织（透明细胞肾细胞癌、结肠癌和胃肠道间质瘤）中，采用qRT-PCR测定了广泛用作参考基因的五种基因（18 S、B2 M、HPRT 1、LMNB 1和SDHA）和两种靶基因（TP 53和MMP 2）的表达。我们证明，一个参考基因在一种肿瘤类型中的稳定表达并不能预测在另一种肿瘤类型中的稳定表达。此外，我们发现即使在一种肿瘤类型中，参考基因的表达对于不同的生物学分组比较也是不稳定的。这些观察结果证实了不存在通用的参考基因，并强调了对任何实验条件下的潜在参考基因进行特异性验证的重要性。(C)2004年爱思唯尔公司All rights reserved.
In quantitative reverse transcription-polymerase chain reaction (qRT-PCR), normalization using reference genes is a common useful approach, but the validation of suitable reference genes remains a crucial problem. Use of unconfirmed reference genes may lead to misinterpretation of the expression of target genes. The aim of this study was to adapt an adequate statistical approach to identify and validate reference genes suitable for normalization in qRT-PCR assays. We introduce the equivalence test for the identification of stably expressed reference genes. To evaluate the advantages of this test, the expression of five genes widely used as reference genes (18S, B2M, HPRT1, LMNB1, and SDHA), and of two target genes (TP53 and MMP2), was determined with qRT-PCR in different tissues (clear cell renal cell carcinoma, colon carcinoma, and gastrointestinal stromal tumors). We demonstrate that a stable expression of a reference gene in one tumor type does not predict a stable expression in another tumor type. In addition, we found that even within one tumor type, the expression of a reference gene was not stable for different biological groupwise comparisons. These observations confirm that there is no universal reference gene and underline the importance of specific validation of potential reference genes for any experimental condition. (C) 2004 Elsevier Inc. All rights reserved.