Cook Naomi L, Vink Robert, Donkin James J, van den Heuvel Corinna
Discipline of Pathology, The University of Adelaide, Adelaide, South Australia, Australia.
J Neurosci Res. 2009 Jan;87(1):34-41. doi: 10.1002/jnr.21846.
Careful validation of reference genes used for the normalization of real-time RT-PCR data is required to obtain accurate results regarding gene expression. We evaluated the stability of seven commonly used reference genes in the cerebral cortex and hippocampus of rats 3 days following traumatic brain injury (TBI). HPRT, SDHA, and GUSB were found to be the most stable reference genes in the cerebral cortex, whereas B2MG, TBP, and GAPDH were the most stable in the hippocampus. The use of three reference genes was determined to be the optimal number for accurate normalization of data. To illustrate this point, when our gene of interest, substance P (SP), was normalized against the three most stable reference genes in both brain areas, we found no significant difference between injured and uninjured rats at the 3-day time point. However, when our SP data were normalized to each reference gene individually, SP mRNA level was highly variable depending on the reference gene chosen. The results of the present study highlight the importance of validating reference genes to be used for real-time RT-PCR analysis. The use of the most stable reference genes presented here will allow more accurate normalization of gene expression data in TBI.
为了获得关于基因表达的准确结果，需要对用于实时逆转录聚合酶链反应（RT-PCR）数据标准化的参考基因进行仔细验证。我们评估了创伤性脑损伤（TBI）后3天大鼠大脑皮层和海马中七个常用参考基因的稳定性。发现次黄嘌呤磷酸核糖转移酶（HPRT）、琥珀酸脱氢酶（SDHA）和β-葡萄糖醛酸酶（GUSB）是大脑皮层中最稳定的参考基因，而β2微球蛋白（B2MG）、TATA盒结合蛋白（TBP）和甘油醛-3-磷酸脱氢酶（GAPDH）是海马中最稳定的参考基因。确定使用三个参考基因是准确标准化数据的最佳数量。为了说明这一点，当我们感兴趣的基因——P物质（SP），相对于两个脑区中最稳定的三个参考基因进行标准化时，我们发现在3天时间点受伤和未受伤的大鼠之间没有显著差异。然而，当我们的SP数据分别针对每个参考基因进行标准化时，SP mRNA水平根据所选参考基因的不同而有很大差异。本研究结果突出了验证用于实时RT-PCR分析的参考基因的重要性。使用这里呈现的最稳定的参考基因将使TBI中基因表达数据的标准化更加准确。
