Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription.
Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription.
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DOI：
10.3969/j.issn.1673-5374.2012.14.004
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发表时间：
2012-05-15
影响因子：
6.1
通讯作者：
Czaja K
中科院分区：
文献类型：
--
作者：
Johnston S;Gallaher Z;Czaja K
实时定量逆转录-聚合酶链反应（qPCR）被广泛用于研究神经系统实验操作后的转录变化。尽管qPCR被广泛使用，但由于内源性转录的动态性质，结果的解释因缺乏合适的参考基因而受到损害。为了解决这一固有缺陷，我们研究了使用外源性的spike-in mRNA，荧光素酶，作为内部参考基因的2-氨基葡萄糖Ct标准化方法。为了诱导动态转录，我们向成年雄性Sprague-Dawley大鼠全身给予辣椒素（一种对表达TRPV-1受体的C型感觉神经元有选择性的神经毒素）。我们随后分离结状神经节用于qPCR分析，参考是外源性荧光素酶mRNA或常用的内源性参考β-III微管蛋白。外源性荧光素酶mRNA参考清楚地证明了内源性参考的动态表达。此外，内源性参考的变异性会导致对其他感兴趣基因的误解。总之，传统的参考基因在生理正常情况下往往是不稳定的，并且在神经系统受损后肯定是不稳定的。外源性加标参比品的使用为qPCR数据分析提供了一致且易于实施的替代方案。
Quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2-∆∆Ct normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxin selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference β-III tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.
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影响因子：
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