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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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Evaluation of the optimum number of reference genes according to the geNorm software. The magnitude of the change in the normalisation factor after the inclusion of an additional reference gene reflects the improvement that is obtained. Vn/n + 1 represents a comparison of the different models, which are those with n and n+1 reference genes.
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Figure 8: Evaluation of the optimum number of reference genes according to the geNorm software. The magnitude of the change in the normalisation factor after the inclusion of an additional reference gene reflects the improvement that is obtained. Vn/n + 1 represents a comparison of the different models, which are those with n and n+1 reference genes.

Mentions: The geNorm analysis also allows for evaluation of the optimal number of reference genes required for a reliable and accurate normalisation of expression data. It is suggested that the Vn/Vn+1 cut-off value of 0.15 should be considered as a limit beneath which the involvement of additional reference genes would not be required [14]. However, based on our experimental data, the variation Vn/Vn+1 did not reach this cut-off; the observed variability ranged from 0.221 for the addition of a third reference gene (n = 2) to 0.798 for the addition of five reference genes (n = 4) (Figure 8). With the increase in the number of genes, the variation Vn/Vn + 1 did not decrease. This may be related to the changes in TUBA1A and VIM.


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)

Evaluation of the optimum number of reference genes according to the geNorm software. The magnitude of the change in the normalisation factor after the inclusion of an additional reference gene reflects the improvement that is obtained. Vn/n + 1 represents a comparison of the different models, which are those with n and n+1 reference genes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Evaluation of the optimum number of reference genes according to the geNorm software. The magnitude of the change in the normalisation factor after the inclusion of an additional reference gene reflects the improvement that is obtained. Vn/n + 1 represents a comparison of the different models, which are those with n and n+1 reference genes.
Mentions: The geNorm analysis also allows for evaluation of the optimal number of reference genes required for a reliable and accurate normalisation of expression data. It is suggested that the Vn/Vn+1 cut-off value of 0.15 should be considered as a limit beneath which the involvement of additional reference genes would not be required [14]. However, based on our experimental data, the variation Vn/Vn+1 did not reach this cut-off; the observed variability ranged from 0.221 for the addition of a third reference gene (n = 2) to 0.798 for the addition of five reference genes (n = 4) (Figure 8). With the increase in the number of genes, the variation Vn/Vn + 1 did not decrease. This may be related to the changes in TUBA1A and VIM.

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