The lack of a systematic validation of reference genes:: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants
The lack of a systematic validation of reference genes:: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants
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DOI：
10.1111/j.1467-7652.2008.00346.x
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发表时间：
2008-08-01
影响因子：
13.8
通讯作者：
Van Wuytswinkel, Olivier
中科院分区：
文献类型：
--
作者：
Gutierrez, Laurent;Mauriat, Melanie;Van Wuytswinkel, Olivier
逆转录-聚合酶链反应（RT-PCR）方法已被用于大比例的转录组分析发表的日期。通过该方法获得的结果的准确性强烈依赖于使用稳定表达的基因（称为参考）的准确转录物标准化。最近已经开发了统计算法来帮助验证参考基因，并且大多数哺乳动物、酵母和细菌中基因表达的研究现在包括这种验证。令人惊讶的是，这种重要的方法在植物研究中没有得到充分利用，在植物研究中，假定的管家基因往往被用作参考，而没有任何适当的验证。采用定量RT-PCR技术，检测了几个常用基因在拟南芥（Arabidopsis thaliana）和杂交白杨（Populus tremula x Populus tremuloides）不同组织中的表达稳定性。结果发现，这些基因的表达大多是不稳定的，表明它们作为参考是不合适的。本研究证明了使用此类不适当参考品对RT-PCR所得结果的主要影响。使用白杨作为一个模型，证据表明，没有基因可以作为一个通用的参考，这意味着需要一个系统的验证参考基因。这是第一次，在何种程度上缺乏一个系统的验证参考基因是一个绊脚石，通过RT-PCR在植物中获得的结果的可靠性清楚地显示。
Reverse transcription-polymerase chain reaction (RT-PCR) approaches have been used in a large proportion of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes, and most studies of gene expression in mammals, yeast and bacteria now include such validation. Surprisingly, this important approach is under-utilized in plant studies, where putative housekeeping genes tend to be used as references without any appropriate validation. Using quantitative RT-PCR, the expression stability of several genes commonly used as references was tested in various tissues of Arabidopsis thaliana and hybrid aspen (Populus tremula x Populus tremuloides). It was found that the expression of most of these genes was unstable, indicating that their use as references is inappropriate. The major impact of the use of such inappropriate references on the results obtained by RT-PCR is demonstrated in this study. Using aspen as a model, evidence is presented indicating that no gene can act as a universal reference, implying the need for a systematic validation of reference genes. For the first time, the extent to which the lack of a systematic validation of reference genes is a stumbling block to the reliability of results obtained by RT-PCR in plants is clearly shown.