Reference genes for quantitative real time PCR in UVB irradiated keratinocytes

Attila Balogh, György Paragh, A. Juhász, Tamás Köbling, D. Törőcsik, Edit Mikó, Viktória Varga, Gabriella Emri, Irén Horkay, B. Scholtz, E. Remenyik

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a sensitive and highly reproducible method often used for determining mRNA levels. To enable proper comparison of gene expression genes expressed at stabile levels within the cells in the studied experimental system need to be identified and used as reference. Ultraviolet B (UVB) radiation is an exogenous carcinogenic stimulus in keratinocytes, and UVB elicited changes have extensively been studied by qRT-PCR, yet a comparison of commonly used reference genes in UVB treatment is lacking. To find the best genes for compensating slight inter-sample variations in keratinocytes in UVB experiments and to understand the potential effects of improper reference gene (RG) selection we have analyzed the mRNA expression of 10 housekeeping genes in neonatal human epidermal keratinocytes (NHEK) after UVB treatment. The biological effect of the used UVB light source was validated by trypane blue exclusion, MTT and comet assays. 20-40 mJ/cm2 dose was chosen for the experiments. The stability of the 10 RGs was assessed by the GeNorm and Normfinder software tools. Regardless of their slightly different algorithm the programs found succinate dehydrogenase complex subunit A (SDHA) to be the best individual RG and SDHA and phosphoglycerate kinase-1 (PGK1) as the most suitable combination. Analysis of the expression of tumor necrosis factor alpha (TNFα) and vascular endothelial growth factor (VEGF) found that while the perception of changes in TNF-alpha, a gene undergoing marked upregulation after UVB irradiation is independent of the used RG, changes seen in the more modestly upregulated VEGF are greatly effected by reference gene selection. These findings highlight the importance of reference gene selection in UVB irradiation experiments, and provide evidence that using SDHA or the combination of SDHA and PGK1 as standards could be a reliable method for normalizing qRT-PCR results in keratinocytes after UVB treatment.

Original languageEnglish
Pages (from-to)133-139
Number of pages7
JournalJournal of Photochemistry and Photobiology, B: Biology
Volume93
Issue number3
DOIs
Publication statusPublished - Dec 11 2008

Fingerprint

Keratinocytes
genes
Real-Time Polymerase Chain Reaction
Succinate Dehydrogenase
dehydrogenases
Genes
polymerase chain reaction
Phosphoglycerate Kinase
Reverse Transcription
necrosis
Polymerase Chain Reaction
Vascular Endothelial Growth Factor A
Tumor Necrosis Factor-alpha
tumors
Messenger RNA
Comet Assay
Essential Genes
normalizing
irradiation
biological effects

Keywords

  • Housekeeping gene
  • Keratinocyte
  • Normalization
  • Real time quantitative PCR
  • Reference
  • UVB

ASJC Scopus subject areas

  • Radiation
  • Radiology Nuclear Medicine and imaging
  • Radiological and Ultrasound Technology
  • Biophysics

Cite this

Reference genes for quantitative real time PCR in UVB irradiated keratinocytes. / Balogh, Attila; Paragh, György; Juhász, A.; Köbling, Tamás; Törőcsik, D.; Mikó, Edit; Varga, Viktória; Emri, Gabriella; Horkay, Irén; Scholtz, B.; Remenyik, E.

In: Journal of Photochemistry and Photobiology, B: Biology, Vol. 93, No. 3, 11.12.2008, p. 133-139.

Research output: Contribution to journalArticle

Balogh, Attila ; Paragh, György ; Juhász, A. ; Köbling, Tamás ; Törőcsik, D. ; Mikó, Edit ; Varga, Viktória ; Emri, Gabriella ; Horkay, Irén ; Scholtz, B. ; Remenyik, E. / Reference genes for quantitative real time PCR in UVB irradiated keratinocytes. In: Journal of Photochemistry and Photobiology, B: Biology. 2008 ; Vol. 93, No. 3. pp. 133-139.
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