Santos Ana Rita A, Duarte Carlos B
Center for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra, Coimbra, Portugal.
J Neurosci Res. 2008 Dec;86(16):3684-92. doi: 10.1002/jnr.21796.
The stability of expression of an internal control is required for accurate and reliable normalization in quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) experiments. However, expression of commonly used reference genes can be regulated under specific experimental conditions, particularly in response to stimuli that exert multiple effects on gene expression. The neurotrophin brain-derived neurotrophic factor (BDNF) regulates gene expression through activation of multiple signaling cascades, and we have conducted an expression study for the proper validation of internal control genes in BDNF-stimulated cultured hippocampal neurons. geNorm and NormFinder were applied to eight potential genes to identify the most stable genes to be used in the relative quantification of the effects of BDNF on gene expression. Our data show that Tbp (TATA box binding protein), Ppia (peptidylprolyl isomerase A), Pgk1 (phosphoglycerate kinase 1), and Hprt1 (hypoxanthine guanine phosphoribosyl transferase I) are the most stable genes under the experimental conditions used, contrasting with Tuba1 (tubulin alpha1-A chain) and Gapdh (glyceraldehydes-3-phosphate dehydrogenase), two genes widely used as control genes, which showed an unstable expression in hippocampal neurons stimulated with BDNF. Analysis of the BDNF-induced changes in expression of Sars, Tufm, and Egr3 by using different sets of control genes showed distinct results, with a combination of three to four of the genes Tbp/Ppia/Pgk1/Hprt1 providing the most consistent results. Our data reinforce the need for proper validation of the internal control genes for an accurate quantification of qRT-PCR results, particularly when analyzing cellular responses to agents (e.g., neurotrophins) that cause multiple changes in gene expression.
在实时定量逆转录-聚合酶链反应（qRT-PCR）实验中，为了进行准确可靠的标准化分析，需要内部控制基因表达稳定。然而，常用参考基因的表达在特定实验条件下可能会受到调控，特别是在对基因表达产生多种影响的刺激作用下。神经营养因子脑源性神经营养因子（BDNF）通过激活多个信号级联反应来调节基因表达，我们进行了一项表达研究，以在BDNF刺激的培养海马神经元中正确验证内部控制基因。将geNorm和NormFinder应用于八个潜在基因，以鉴定用于相对定量BDNF对基因表达影响的最稳定基因。我们的数据表明，在所使用的实验条件下，Tbp（TATA盒结合蛋白）、Ppia（肽基脯氨酰异构酶A）、Pgk1（磷酸甘油酸激酶1）和Hprt1（次黄嘌呤鸟嘌呤磷酸核糖基转移酶I）是最稳定的基因，这与广泛用作对照基因的Tuba1（微管蛋白α1-A链）和Gapdh（甘油醛-3-磷酸脱氢酶）形成对比，它们在BDNF刺激的海马神经元中表现出不稳定的表达。使用不同组对照基因分析BDNF诱导的Sars、Tufm和Egr3表达变化，结果显示出明显差异，Tbp/Ppia/Pgk1/Hprt1这三到四个基因的组合提供了最一致的结果。我们的数据进一步强调，为了准确量化qRT-PCR结果，特别是在分析细胞对引起基因表达多种变化的试剂（如神经营养因子）的反应时 , 需要对内部控制基因进行适当验证。
