Transcriptional expression study in the central nervous system of rats: what gene should be used as internal control?
Transcriptional expression study in the central nervous system of rats: what gene should be used as internal control?
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DOI：
10.1590/s1679-45082014ao3042
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发表时间：
2014-09
期刊：
影响因子：
--
通讯作者：
Veiga AB
中科院分区：
文献类型：
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作者：
Moura AC;Lazzari VM;Agnes G;Almeida S;Giovenardi M;Veiga AB
越来越多发表的文章报道了大鼠中具有不同行为模式的特定基因的表达。信使核糖核酸转录物的水平通常通过逆转录和聚合酶链反应进行分析，并在内参基因（内参基因）归一化后定量。然而，看家基因在中枢神经系统中表现出不同的表达，这取决于生理条件和待研究的大脑区域。选择良好的内控基因是获得可靠结果的关键。本研究评估了三种看家基因（β -肌动蛋白、亲环蛋白A和泛素C）在大鼠中枢神经系统不同区域（嗅球、海马、纹状体和前额皮质）的表达。采用初泌期Wistar大鼠（雌性，n=6）。从大脑的每个区域提取总核糖核酸；通过反转录合成互补脱氧核糖核酸，并使用SYBR™Green和每个内参基因特异性引物进行实时定量聚合酶链反应扩增。使用NormFinder确定表达式的稳定性。β -肌动蛋白是海马和纹状体中最稳定的基因，而亲环蛋白A和泛素C分别在前额皮质和嗅球中表现出更大的稳定性。根据我们的研究，进一步以大鼠为动物模型的基因表达研究，在选择可靠的内控基因时，应该考虑到这些结果。
A growing number of published articles report the expression of specific genes with different behavior patterns in rats. The levels of messenger ribonucleic acid transcripts are usually analyzed by reverse transcription followed by polymerase chain reaction and quantified after normalization with an internal control or reference gene (housekeeping gene). Nevertheless, housekeeping genes exhibit different expression in the central nervous system, depending on the physiological conditions and the area of the brain to be studied. The choice of a good internal control gene is essential for obtaining reliable results. This study evaluated the expression of three housekeeping genes (beta-actin, cyclophilin A, and ubiquitin C) in different areas of the central nervous system in rats (olfactory bulb, hippocampus, striatum, and prefrontal cortex). Wistar rats (virgin females, n=6) during the diestrum period were used. Total ribonucleic acid was extracted from each region of the brain; the complementary deoxyribonucleic acid was synthesized by reverse transcription and amplified by real-time quantitative polymerase chain reaction using SYBR™ Green and primers specific for each one of the reference genes. The stability of the expression was determined using NormFinder. Beta-actin was the most stable gene in the hippocampus and striatum, while cyclophilin A and ubiquitin C showed greater stability in the prefrontal cortex and the olfactory bulb, respectively. Based on our study, further studies of gene expression using rats as animal models should take into consideration these results when choosing a reliable internal control gene.