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

Li Li,Yan Yan,Tao Qu,Baoxi Wang,Haoxiang Xu

doi:10.1186/1471-2199-12-8

Abstract

BackgroundReference 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.ResultsQuantitative 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.ConclusionThe 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.

Highlights

Reference genes are frequently used to normalise mRNA levels between different samples
PCR results in a bright, specific band for the vimentin primers but no specific products for the keratin10 primers by 2% agarose gel electrophoresis analysis
Fibre bundles in the cytoplasm along the cell axis stained green, while the nucleus stained orange by fluorescent immunohistochemical analysis in fibroblasts(Figure 2). These same fibroblasts were negative for the expression of the keratin protein. These results indicated that highly pure human dermal fibroblasts were successfully obtained from the foreskin samples

Summary

Introduction

Reference genes are frequently used to normalise mRNA levels between different samples. Our previous studies have demonstrated that the expression of vimentin, one cytoskeletal protein, was increased in ultraviolet B (UVB)-irradiated fibroblasts. 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. UVB is suspected of lowering the immune defence system of the skin [3] Given these effects, the gene expression of dermal fibroblasts after UVB irradiation has become a significant area of study, Quantitative real-time PCR (qPCR) is the most powerful method used to quantify gene expression. Similar to other expression study methods, the sample data are usually required to be normalised against either another data set or particular references to correct for any differences in the amount of starting material. Cytoskeletal protein genes, including ACTB, TUBA1A (tubulin, alpha 1a), and TUBB1 (tubulin, beta 1), have been used as reference genes for many experimental conditions

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