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Selection of reference genes for gene expression studies in ultraviolet B-irradiated human skin fibroblasts using quantitative real-time PCR.

Li L, Yan Y, Xu H, Qu T, Wang B - BMC Mol. Biol. (2011)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Dermatology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, PR China.

ABSTRACT

Background: 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.

Results: 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/cm(2) and that the expression of TUBA1A was significantly reduced by UVB doses ≥75 mJ/cm(2) 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.

Conclusion: 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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The relative fold expression of VIM in fibroblasts irradiated by UVB at the indicated time points. (Figure 4-A) VIM expression in fibroblasts 24 h after treatment with different does of UVB, including 0, 25, 50, 75, and 100 mJ/cm2. (Figure 4-B) The time course of VIM expression after treatment with 125 mJ/cm2 UVB. The error bars represent the standard deviation. The experiments were performed in triplicate. * p < 0.05 compared with the control groups; # p < 0.05 compared with the group irradiated with a dose of 50 mJ/cm2; Δ p < 0.05 compared with the group 24 h after exposure.
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Figure 4: The relative fold expression of VIM in fibroblasts irradiated by UVB at the indicated time points. (Figure 4-A) VIM expression in fibroblasts 24 h after treatment with different does of UVB, including 0, 25, 50, 75, and 100 mJ/cm2. (Figure 4-B) The time course of VIM expression after treatment with 125 mJ/cm2 UVB. The error bars represent the standard deviation. The experiments were performed in triplicate. * p < 0.05 compared with the control groups; # p < 0.05 compared with the group irradiated with a dose of 50 mJ/cm2; Δ p < 0.05 compared with the group 24 h after exposure.

Mentions: To determine whether UVB affects the expression of cytoskeletal genes in cultured human fibroblasts, the fibroblasts were exposed to various doses of UVB (25, 50, 75, and 100 mJ/cm2). After UVB irradiation, the fibroblasts were cultured for 24 h, and then total RNA was extracted from the cells. qPCR was performed to determine the changes in the expression of these cytoskeletal genes. As common reference genes, the expression levels of ACTB, GAPDH and TUBB1 were not significantly different between the normal and irradiated fibroblasts (data not shown). Doses of 25 and 50 mJ/cm2 did not alter the expression levels of TUBA1A (Figure 3-A), but they were significantly reduced at doses ≥75 mJ/cm2 (P < 0.05). The expression of VIM began to be significantly reduced after exposure to a UVB dose of 25 mJ/cm2 (P < 0.05; Figure 4-A). The decreased expression of VIM became even more pronounced at doses of 75 and 100 mJ/cm2 when compared with 25 and 50 mJ/cm2 (P < 0.05). The mRNA expression levels of TUBA1A and VIM were reduced by varied UVB intensities.


Selection of reference genes for gene expression studies in ultraviolet B-irradiated human skin fibroblasts using quantitative real-time PCR.

Li L, Yan Y, Xu H, Qu T, Wang B - BMC Mol. Biol. (2011)

The relative fold expression of VIM in fibroblasts irradiated by UVB at the indicated time points. (Figure 4-A) VIM expression in fibroblasts 24 h after treatment with different does of UVB, including 0, 25, 50, 75, and 100 mJ/cm2. (Figure 4-B) The time course of VIM expression after treatment with 125 mJ/cm2 UVB. The error bars represent the standard deviation. The experiments were performed in triplicate. * p < 0.05 compared with the control groups; # p < 0.05 compared with the group irradiated with a dose of 50 mJ/cm2; Δ p < 0.05 compared with the group 24 h after exposure.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3050716&req=5

Figure 4: The relative fold expression of VIM in fibroblasts irradiated by UVB at the indicated time points. (Figure 4-A) VIM expression in fibroblasts 24 h after treatment with different does of UVB, including 0, 25, 50, 75, and 100 mJ/cm2. (Figure 4-B) The time course of VIM expression after treatment with 125 mJ/cm2 UVB. The error bars represent the standard deviation. The experiments were performed in triplicate. * p < 0.05 compared with the control groups; # p < 0.05 compared with the group irradiated with a dose of 50 mJ/cm2; Δ p < 0.05 compared with the group 24 h after exposure.
Mentions: To determine whether UVB affects the expression of cytoskeletal genes in cultured human fibroblasts, the fibroblasts were exposed to various doses of UVB (25, 50, 75, and 100 mJ/cm2). After UVB irradiation, the fibroblasts were cultured for 24 h, and then total RNA was extracted from the cells. qPCR was performed to determine the changes in the expression of these cytoskeletal genes. As common reference genes, the expression levels of ACTB, GAPDH and TUBB1 were not significantly different between the normal and irradiated fibroblasts (data not shown). Doses of 25 and 50 mJ/cm2 did not alter the expression levels of TUBA1A (Figure 3-A), but they were significantly reduced at doses ≥75 mJ/cm2 (P < 0.05). The expression of VIM began to be significantly reduced after exposure to a UVB dose of 25 mJ/cm2 (P < 0.05; Figure 4-A). The decreased expression of VIM became even more pronounced at doses of 75 and 100 mJ/cm2 when compared with 25 and 50 mJ/cm2 (P < 0.05). The mRNA expression levels of TUBA1A and VIM were reduced by varied UVB intensities.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Dermatology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, PR China.

ABSTRACT

Background: 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.

Results: 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/cm(2) and that the expression of TUBA1A was significantly reduced by UVB doses ≥75 mJ/cm(2) 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus