Selection of reference genes for gene expression studies in ultraviolet B-irradiated human skin fibroblasts using quantitative real-time PCR.
Selection of reference genes for gene expression studies in ultraviolet B-irradiated human skin fibroblasts using quantitative real-time PCR.
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DOI：
10.1186/1471-2199-12-8
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发表时间：
2011-02-17
影响因子：
--
通讯作者：
Wang B
中科院分区：
文献类型：
--
作者：
Li L;Yan Y;Xu H;Qu T;Wang B
参考基因经常被用来使不同样本之间的mRNA水平正常化。然而，这些基因的表达水平可能会因组织或细胞的不同而不同，并且在某些情况下可能会改变。细胞骨架基因作为参考基因已成为实验研究的多功能工具。我们以前的研究已经证明，在紫外线B(UVB)照射的成纤维细胞中，细胞骨架蛋白Vimentin的表达增加。因此，我们检测了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，以确定这些候选基因中哪些是最合适的参考基因。用NormFinder和geNorm软件程序进行定量实时聚合酶链式反应分析。表达谱的初步筛选表明，≥剂量为25mJ/cm~2的中波紫外线可抑制培养的人真皮成纤维细胞中VIM的表达，而≥剂量为75mJ/cm~2的UVB可显著降低人真皮成纤维细胞中VIM的表达。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(甘油醛-3-磷酸脱氢酶)。相比之下，vim被发现是最不稳定的基因。ACTB和TUBB1的组合被揭示为在数据归一化中引入最小系统误差的基因对。这些结果证明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，因此在任何涉及UVB照射的人皮肤成纤维细胞的基因表达研究中都应该排除它们作为参考基因。
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.
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DOI：
10.1111/j.1600-0781.2010.00522.x
发表时间：
2010-08-01
影响因子：
2.6
作者：
Xu, Haoxiang;Yan, Yan;Wang, Baoxi
通讯作者：
Wang, Baoxi
影响因子：
--
作者：
Jung M;Ramankulov A;Roigas J;Johannsen M;Ringsdorf M;Kristiansen G;Jung K
通讯作者：
Jung K
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
2.9
作者：
Johansson, Sofia;Fuchs, Andrea;Ekstrom, Tomas J.
通讯作者：
Ekstrom, Tomas J.
影响因子：
10
作者：
Glare, EM;Divjak, M;Walters, EH
通讯作者：
Walters, EH