Comparison of in vitro and in vivo reference genes for internal standardization of real-time PCR data
Comparison of in vitro and in vivo reference genes for internal standardization of real-time PCR data
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DOI：
10.2144/000112052
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发表时间：
2006-02-01
期刊：
影响因子：
2.7
通讯作者：
Brüss, M
中科院分区：
文献类型：
--
作者：
Gilsbach, R;Kouta, M;Brüss, M
实时 PCR 是一种强大的基因表达研究技术，在许多临床和科学领域变得越来越重要。所获得结果的显着性很大程度上取决于数据的标准化以补偿样本之间的差异。最广泛使用的方法是使用内源参考基因（管家基因）作为内标。这种方法在文献中存在争议，因为没有一个参考基因在所有生物样品中稳定表达。因此，必须针对每个实验条件验证候选参考基因。在我们的研究中，我们引入并评估了体外合成的参考 cRNA，用于相对信使 RNA (mRNA) 表达模式的内部标准化。该参考文献由水母蛋白（一种水母蛋白）的体外转录编码序列组成，被纳入提取的 RNA 中。该方法的实验意义通过小鼠大脑不同区域中神经营养素 3 mRNA 的表达进行了代表性测试。与三个稳定表达的参考基因 [β-肌动蛋白、甘油醛-3-磷酸脱氢酶 (GAPDH) 和次黄嘌呤磷酸核糖基转移酶 1 (HPRT1)] 的比较证明，模板 RNA 与体外转录的 cRNA 的尖峰是实时 PCR 实验内部标准化的宝贵工具。
Real-time PCR is a powerful technique for gene expression studies, which have become increasingly important in a large number of clinical and scientific fields. The significance of the obtained results strongly depends on the normalization of the data to compensate for differences between the samples. The most widely used approach is to use endogenous reference genes (housekeeping genes) as internal standards. This approach is controversially discussed in the literature because none of the reference genes is stably expressed throughout all biological samples. Therefore, candidate reference genes have to be validated for each experimental condition. In our studies, we introduced and evaluated an in vitro synthesized reference cRNA for internal standardization of relative messenger RNA (mRNA) expression patterns. This reference, consisting of the in vitro transcribed coding sequence of aequorin, a jellyfish protein, was incorporated in the extracted RNA. The experimental significance of this approach was representatively tested for the expression of the neurotrophin-3 mRNA in distinct regions of mouse brains. A comparison to three stably expressed reference genes [beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine phosphoribosyl-transferase 1 (HPRT1)] gave evidence that the spiking of template RNA with in vitro transcribed cRNA is a valuable tool for internal standardization of real-time PCR experiments.