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首页> 外文期刊>BMC Molecular Biology >Selection of reference genes for gene expression studies in ultraviolet B-irradiated human skin fibroblasts using quantitative real-time PCR
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Selection of reference genes for gene expression studies in ultraviolet B-irradiated human skin fibroblasts using quantitative real-time PCR

机译：使用实时荧光定量PCR选择紫外线B照射的人皮肤成纤维细胞中用于基因表达研究的参考基因

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Reference genes are frequently used to normalise mRNA levels between different samples. The expression level of these genes, however, may vary between tissues or cells and may change under certain circumstances. Cytoskeleton genes have served as multifunctional tools for experimental studies as reference genes. Our previous studies have demonstrated that the expression of vimentin, one cytoskeletal protein, was increased in ultraviolet B (UVB)-irradiated fibroblasts. Thus, we examined the expression of other cytoskeleton protein genes, ACTB (actin, beta), TUBA1A (tubulin, alpha 1a), and TUBB1 (tubulin, beta 1), in human dermal fibroblasts irradiated by UVB to determine which of these candidates were the most appropriate reference genes. Quantitative real-time PCR followed by analysis with the NormFinder and geNorm software programmes was performed. The initial screening of the expression patterns demonstrated that the expression of VIM was suppressed after UVB irradiation at doses ≥25 mJ/cm2 and that the expression of TUBA1A was significantly reduced by UVB doses ≥75 mJ/cm2 in cultured human dermal fibroblasts. The analysis of the experimental data revealed ACTB to be the most stably expressed gene, followed by GAPDH (aglyceraldehyde-3-phosphate dehydrogenase), under these experimental conditions. By contrast, VIM was found to be the least stable gene. The combination of ACTB and TUBB1 was revealed to be the gene pair that introduced the least systematic error into the data normalisation. The data herein provide evidence that ACTB and TUBB1 are suitable reference genes in human skin fibroblasts irradiated by UVB, whereas VIM and TUBA1A are not and should therefore be excluded as reference genes in any gene expression studies involving UVB-irradiated human skin fibroblasts.
机译：参考基因经常用于标准化不同样品之间的mRNA水平。但是，这些基因的表达水平在组织或细胞之间可能会有所不同，并且在某些情况下可能会发生变化。细胞骨架基因已作为参考基因作为实验研究的多功能工具。我们以前的研究表明，波形蛋白（一种细胞骨架蛋白）的表达在紫外线B（UVB）照射的成纤维细胞中增加。因此，我们检查了UVB照射的人真皮成纤维细胞中其他细胞骨架蛋白基因ACTB（肌动蛋白，β），TUBA1A（微管蛋白，α1a）和TUBB1（微管蛋白，β1）的表达，以确定其中哪些是最合适的参考基因。进行定量实时PCR，然后使用NormFinder和geNorm软件程序进行分析。表达模式的初步筛选表明，在≥25 mJ / cm2的剂量的UVB照射后，VIM的表达受到抑制，而在≥75mJ / cm2的UVB剂量下，培养的人皮肤成纤维细胞中TUBA1A的表达显着降低。对实验数据的分析表明，在这些实验条件下，ACTB是最稳定表达的基因，其次是GAPDH（3-磷酸甘油醛脱氢酶）。相反，发现VIM是最不稳定的基因。 ACTB和TUBB1的组合被发现是在数据标准化中引入最小系统误差的基因对。本文中的数据提供了证据，表明ACTB和TUBB1是UVB照射的人皮肤成纤维细胞中合适的参考基因，而VIM和TUBA1A不是，因此在涉及UVB照射的人皮肤成纤维细胞的任何基因表达研究中均应排除VIM和TUBA1A作为参考基因。

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