Background

The genes encoding β-actin and GAPDH are two of the most commonly used reference genes for normalization in in vitro blood-brain barrier studies. Studies have, however, shown that these reference genes might not always be the best choice. The aim of the present study was to evaluate 10 reference genes for use in mRNA profiling studies in primary cultures of brain endothelial cells of bovine origin.

Methods

Gene evaluations were performed by qPCR in mono-culture and in co-cultures with astrocytes. The expression of reference genes was furthermore investigated during culture. Qbase+ software was used to analyze the stability of the tested genes and for determinations of the optimal number of reference genes.

Results

The stability of the reference genes varied between the culture configurations, but for all culture configurations we found that the optimal number of reference genes were two. PMM-1, RPL13A and β-actin were the most stable genes in mono-cultures, non-contact co-culture and contact co-culture respectively. For studies comparing gene expression between different culture configurations the optimal number of reference genes was three and RPL13A was found to be most stable. During cell culture a number of four reference genes were found to be optimal and YWHAZ was found to be the most stable gene. β-actin and GAPDH were found to be the least stable genes during culture.

Conclusion

Overall we found that the validation of reference genes was important in order to normalize target gene expression correctly, and suggest sets of reference genes to be used under different experimental conditions, in order to quantify mRNA transcript levels in blood-brain barrier cell models correctly.

中文翻译：

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验证参考基因以标准化实时定量PCR研究体外培养的脑毛细血管内皮细胞中的基因表达

背景

编码β-肌动蛋白和GAPDH的基因是体外血脑屏障研究中最常用于标准化的两个参考基因。但是研究表明，这些参考基因可能并不总是最佳选择。本研究的目的是评估在牛源性脑内皮细胞原代培养物中用于mRNA谱分析研究的10个参考基因。

方法

通过qPCR在星形胶质细胞的单培养和共培养中进行基因评估。在培养过程中还研究了参考基因的表达。使用Qbase +软件分析测试基因的稳定性并确定参考基因的最佳数量。

结果

参考基因的稳定性在不同的培养配置之间有所不同，但是对于所有培养配置，我们发现参考基因的最佳数量为两个。PMM-1，RPL13A和β-肌动蛋白分别是单培养，非接触共培养和接触共培养中最稳定的基因。对于比较不同培养物配置之间的基因表达的研究，参考基因的最佳数目为3，并且发现RPL13A最稳定。在细胞培养过程中，发现有四个参考基因是最佳基因，而YWHAZ被发现是最稳定的基因。发现β-肌动蛋白和GAPDH是培养过程中最不稳定的基因。

结论

总体而言，我们发现参考基因的验证对于正确正确地标准化靶基因表达非常重要，并建议在不同实验条件下使用的参考基因组，以正确定量血脑屏障细胞模型中的mRNA转录水平。