Reference gene validation for qPCR on normoxia- and hypoxia-cultured human dermal fibroblasts exposed to UVA: Is β-actin a reliable normalizer for photoaging studies?

Journal of Biotechnology

Volumn 156, Issue 3, 2011, Pages 153-162

  Bruge F ^a   Venditti, E ^a   Tiano, L ^a   Littarru, G P ^a   Damiani, E ^a  

^a UNIVERSITÀ POLITECNICA DELLE MARCHE   (Italy)

Author keywords

Human dermal fibroblasts; Hypoxia normoxia; Normalization; Reference genes; RT qPCR; UVA

Indexed keywords

HUMAN DERMAL FIBROBLASTS; HYPOXIA/NORMOXIA; NORMALIZATION; REFERENCE GENES; RT-QPCR; UVA;

CELL CULTURE; FIBROBLASTS; GENES; OXYGEN; POLYMERASE CHAIN REACTION; PROTEINS;

TRANSCRIPTION;

BETA ACTIN; MESSENGER RNA;

ARTICLE; CELL VIABILITY; CONTROLLED STUDY; FIBROBLAST; GENE EXPRESSION; HUMAN; HUMAN CELL; HYPOXIA; NUCLEOTIDE SEQUENCE; OXYGEN TENSION; PHOTOAGING; POLYMERASE CHAIN REACTION; PRIORITY JOURNAL; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; ULTRAVIOLET A RADIATION;

ACTINS; CELL HYPOXIA; CELLS, CULTURED; COLLAGEN TYPE I; FIBROBLASTS; GENE EXPRESSION PROFILING; HUMANS; MATRIX METALLOPROTEINASE 1; REAL-TIME POLYMERASE CHAIN REACTION; REFERENCE STANDARDS; REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION; SKIN; ULTRAVIOLET RAYS;

EID: 80054094641     PISSN: 01681656     EISSN: 18734863     Source Type: Journal    
DOI: 10.1016/j.jbiotec.2011.09.018     Document Type: Article

References (60)

1. Andersen C.L., Jensen J.L., Orntoft T.F. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004, 64:5245-5250.
2. Bae J.T., Sim G.S., Kim J.H., Pyo H.B., Yun J.W., Lee B.C. Antioxidative activity of the hydrolytic enzyme treated Sorbus commixta Hedl. and its inhibitory effect on matrix metalloproteinase-1 in UV irradiated human dermal fibroblasts. Arch. Pharm. Res. 2007, 30:1116-1123.
3. Balogh A., Paragh G., Juhasz A., Kobling T., Torocsik D., Miko E., Varga V., Emri G., Horkay I., Scholtz B., Remenyik E. Reference genes for quantitative real time PCR in UVB irradiated keratinocytes. J. Photochem. Photobiol. B 2008, 93:133-139.
4. Bas A., Forsberg G., Hammarstrom S., Hammarstrom M.L. Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes. Scand. J. Immunol. 2004, 59:566-573.
5. Besaratinia A., Kim S.I., Pfeifer G.P. Rapid repair of UVA-induced oxidized purines and persistence of UVB-induced dipyrimidine lesions determine the mutagenicity of sunlight in mouse cells. FASEB J. 2008, 22:2379-2392.
6. Bickers D.R., Athar M. Oxidative stress in the pathogenesis of skin disease. J. Invest. Dermatol. 2006, 126:2565-2575.
7. Boraldi F., Annovi G., Carraro F., Naldini A., Tiozzo R., Sommer P., Quaglino D. Hypoxia influences the cellular cross-talk of human dermal fibroblasts. A proteomic approach. Biochim. Biophys. Acta 2007, 1774:1402-1413.
8. Buechner N., Schroeder P., Jakob S., Kunze K., Maresch T., Calles C., Krutmann J., Haendeler J. Changes of MMP-1 and collagen type Ialpha1 by UVA, UVB and IRA are differentially regulated by Trx-1. Exp. Gerontol. 2008, 43:633-637.
9. Cheng S., Tada M., Hida Y., Asano T., Kuramae T., Takemoto N., Hamada J., Miyamoto M., Hirano S., Kondo S., Moriuchi T. High MMP-1 mRNA expression is a risk factor for disease-free and overall survivals in patients with invasive breast carcinoma. J. Surg. Res. 2008, 146:104-109.
10. Chey S., Claus C., Liebert U.G. Validation and application of normalization factors for gene expression studies in Rubella virus-infected cell lines with quantitative real-time PCR. J. Cell. Biochem. 2010, 110:118-128.
11. Cho S., Lee M.J., Kim M.S., Lee S., Kim Y.K., Lee D.H., Lee C.W., Cho K.H., Chung J.H. Infrared plus visible light and heat from natural sunlight participate in the expression of MMPs and type I procollagen as well as infiltration of inflammatory cell in human skin in vivo. J. Dermatol. Sci. 2008, 50:123-133.
12. Cho Y.H., Kim J.H., Sim G.S., Lee B.C., Pyo H.B., Park H.D. Inhibitory effects of antioxidant constituents from Melothria heterophylla on matrix metalloproteinase-1 expression in UVA-irradiated human dermal fibroblasts. J. Cosmet. Sci. 2006, 57:279-289.
13. Creighton-Gutteridge M., Tyrrell R.M. A novel iron chelator that does not induce HIF-1 activity. Free Radic. Biol. Med. 2002, 33:356-363.
14. de Gruijl F.R. Photocarcinogenesis: UVA vs UVB. Methods Enzymol. 2000, 319:359-366.
15. Derveaux S., Vandesompele J., Hellemans J. How to do successful gene expression analysis using real-time PCR. Methods 2010, 50:227-230.
16. Dheda K., Huggett J.F., Chang J.S., Kim L.U., Bustin S.A., Johnson M.A., Rook G.A., Zumla A. The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal. Biochem. 2005, 344:141-143.
17. Falanga V., Martin T.A., Takagi H., Kirsner R.S., Helfman T., Pardes J., Ochoa M.S. Low oxygen tension increases mRNA levels of alpha 1 (I) procollagen in human dermal fibroblasts. J. Cell. Physiol. 1993, 157:408-412.
18. Falanga V., Zhou L., Yufit T. Low oxygen tension stimulates collagen synthesis and COL1A1 transcription through the action of TGF-beta1. J. Cell. Physiol. 2002, 191:42-50.
19. Fligiel S.E., Varani J., Datta S.C., Kang S., Fisher G.J., Voorhees J.J. Collagen degradation in aged/photodamaged skin in vivo and after exposure to matrix metalloproteinase-1 in vitro. J. Invest. Dermatol. 2003, 120:842-848.
20. Foldager C.B., Munir S., Ulrik-Vinther M., Soballe K., Bunger C., Lind M. Validation of suitable house keeping genes for hypoxia-cultured human chondrocytes. BMC Mol. Biol. 2009, 10:94.
21. Goidin D., Mamessier A., Staquet M.J., Schmitt D., Berthier-Vergnes O. Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations. Anal. Biochem. 2001, 295:17-21.
22. Halliwell B. Oxidative stress in cell culture: an under-appreciated problem?. FEBS Lett. 2003, 540:3-6.
23. Hantke B., Lahmann C., Venzke K., Fischer T., Kocourek A., Windsor L.J., Bergemann J., Stab F., Tschesche H. Influence of flavonoids and vitamins on the MMP- and TIMP-expression of human dermal fibroblasts after UVA irradiation. Photochem. Photobiol. Sci. 2002, 1:826-833.
24. Heid C.A., Stevens J., Livak K.J., Williams P.M. Real time quantitative PCR. Genome Res. 1996, 6:986-994.
25. Herrick S.E., Ireland G.W., Simon D., McCollum C.N., Ferguson M.W. Venous ulcer fibroblasts compared with normal fibroblasts show differences in collagen but not fibronectin production under both normal and hypoxic conditions. J. Invest. Dermatol. 1996, 106:187-193.
26. Jenkins G. Molecular mechanisms of skin ageing. Mech. Ageing Dev. 2002, 123:801-810.
27. Kan C., Abe M., Yamanaka M., Ishikawa O. Hypoxia-induced increase of matrix metalloproteinase-1 synthesis is not restored by reoxygenation in a three-dimensional culture of human dermal fibroblasts. J. Dermatol. Sci. 2003, 32:75-82.
28. Karsenty G., Park R.W. Regulation of type I collagen genes expression. Int. Rev. Immunol. 1995, 12:177-185.
29. Krutmann J. The role of UVA rays in skin aging. Eur. J. Dermatol. 2001, 11:170-171.
30. Lee J.H., Chung J.H., Cho K.H. The effects of epigallocatechin-3-gallate on extracellular matrix metabolism. J. Dermatol. Sci. 2005, 40:195-204.
31. Maines M.D., Costa L.G., Reed D.J., Sassa S., Sipes I.G. Current Protocols in Toxicology 1998, John Wiley & Sons, New York.
32. Masek T., Vopalensky V., Suchomelova P., Pospisek M. Denaturing RNA electrophoresis in TAE agarose gels. Anal. Biochem. 2005, 336:46-50.
33. Matrisian L.M. Metalloproteinases and their inhibitors in matrix remodeling. Trends Genet. 1990, 6:121-125.
34. Obermuller-Jevic U.C., Francz P.I., Frank J., Flaccus A., Biesalski H.K. Enhancement of the UVA induction of haem oxygenase-1 expression by beta-carotene in human skin fibroblasts. FEBS Lett. 1999, 460:212-216.
35. Offord E.A., Gautier J.C., Avanti O., Scaletta C., Runge F., Kramer K., Applegate L.A. Photoprotective potential of lycopene, beta-carotene, vitamin E, vitamin C and carnosic acid in UVA-irradiated human skin fibroblasts. Free Radic. Biol. Med. 2002, 32:1293-1303.
36. Philips N., Keller T., Hendrix C., Hamilton S., Arena R., Tuason M., Gonzalez S. Regulation of the extracellular matrix remodeling by lutein in dermal fibroblasts, melanoma cells, and ultraviolet radiation exposed fibroblasts. Arch. Dermatol. Res. 2007, 299:373-379.
37. Philips N., Smith J., Keller T., Gonzalez S. Predominant effects of Polypodium leucotomos on membrane integrity, lipid peroxidation, and expression of elastin and matrixmetalloproteinase-1 in ultraviolet radiation exposed fibroblasts, and keratinocytes. J. Dermatol. Sci. 2003, 32:1-9.
38. Polte T., Tyrrell R.M. Involvement of lipid peroxidation and organic peroxides in UVA-induced matrix metalloproteinase-1 expression. Free Radic. Biol. Med. 2004, 36:1566-1574.
39. Sarasin A. The molecular pathways of ultraviolet-induced carcinogenesis. Mutat. Res. 1999, 428:5-10.
40. Scharffetter-Kochanek K., Brenneisen P., Wenk J., Herrmann G., Ma W., Kuhr L., Meewes C., Wlaschek M. Photoaging of the skin from phenotype to mechanisms. Exp. Gerontol. 2000, 35:307-316.
41. Scharffetter-Kochanek K., Wlaschek M., Brenneisen P., Schauen M., Blaudschun R., Wenk J. UV-induced reactive oxygen species in photocarcinogenesis and photoaging. Biol. Chem. 1997, 378:1247-1257.
42. Scharffetter K., Wlaschek M., Hogg A., Bolsen K., Schothorst A., Goerz G., Krieg T., Plewig G. UVA irradiation induces collagenase in human dermal fibroblasts in vitro and in vivo. Arch. Dermatol. Res. 1991, 283:506-511.
43. Serrano M., Blasco M.A. Putting the stress on senescence. Curr. Opin. Cell Biol. 2001, 13:748-753.
44. Sitkovsky M., Lukashev D. Regulation of immune cells by local-tissue oxygen tension: HIF1 alpha and adenosine receptors. Nat. Rev. Immunol. 2005, 5:712-721.
45. Stucker M., Struk A., Altmeyer P., Herde M., Baumgartl H., Lubbers D.W. The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis. J. Physiol. 2002, 538:985-994.
46. Suganuma K., Nakajima H., Ohtsuki M., Imokawa G. Astaxanthin attenuates the UVA-induced up-regulation of matrix-metalloproteinase-1 and skin fibroblast elastase in human dermal fibroblasts. J. Dermatol. Sci. 2010, 58:136-142.
47. Thellin O., Zorzi W., Lakaye B., De Borman B., Coumans B., Hennen G., Grisar T., Igout A., Heinen E. Housekeeping genes as internal standards: use and limits. J. Biotechnol. 1999, 75:291-295.
48. Toussaint O., Weemaels G., Debacq-Chainiaux F., Scharffetter-Kochanek K., Wlaschek M. Artefactual effects of oxygen on cell culture models of cellular senescence and stem cell biology. J. Cell. Physiol. 2011, 226:315-321.
49. Tricarico C., Pinzani P., Bianchi S., Paglierani M., Distante V., Pazzagli M., Bustin S.A., Orlando C. Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal. Biochem. 2002, 309:293-300.
50. Vandesompele J., De Preter K., Pattyn F., Poppe B., Van Roy N., De Paepe A., Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002, 3. RESEARCH0034.
51. Vandesompele J., Kubista M., Pfaffl M.W. Reference gene validation software for improved normalization. Real-Time PCR Current Technology and Applications 2009, 47-64. Caister Academic Press, Norfolk. J. Logan, K. Edwards, N. Saunders (Eds.).
52. Vielhaber G., Grether-Beck S., Koch O., Johncock W., Krutmann J. Sunscreens with an absorption maximum of >or =360nm provide optimal protection against UVA1-induced expression of matrix metalloproteinase-1, interleukin-1, and interleukin-6 in human dermal fibroblasts. Photochem. Photobiol. Sci. 2006, 5:275-282.
53. Vile G.F., Tyrrell R.M. UVA radiation-induced oxidative damage to lipids and proteins in vitro and in human skin fibroblasts is dependent on iron and singlet oxygen. Free Radic. Biol. Med. 1995, 18:721-730.
54. Webster K.A., Gunning P., Hardeman E., Wallace D.C., Kedes L. Coordinate reciprocal trends in glycolytic and mitochondrial transcript accumulations during the in vitro differentiation of human myoblasts. J. Cell. Physiol. 1990, 142:566-573.
55. Wlaschek M., Briviba K., Stricklin G.P., Sies H., Scharffetter-Kochanek K. Singlet oxygen may mediate the ultraviolet A-induced synthesis of interstitial collagenase. J. Invest. Dermatol. 1995, 104:194-198.
56. Wlaschek M., Wenk J., Brenneisen P., Briviba K., Schwarz A., Sies H., Scharffetter-Kochanek K. Singlet oxygen is an early intermediate in cytokine-dependent ultraviolet-A induction of interstitial collagenase in human dermal fibroblasts in vitro. FEBS Lett. 1997, 413:239-242.
57. Wondrak G.T., Roberts M.J., Cervantes-Laurean D., Jacobson M.K., Jacobson E.L. Proteins of the extracellular matrix are sensitizers of photo-oxidative stress in human skin cells. J. Invest. Dermatol. 2003, 121:578-586.
58. Yamanaka M., Ishikawa O. Hypoxic conditions decrease the mRNA expression of proalpha1(I) and (III) collagens and increase matrix metalloproteinases-1 of dermal fibroblasts in three-dimensional cultures. J. Dermatol. Sci. 2000, 24:99-104.
59. Yan S.X., Hong X.Y., Hu Y., Liao K.H. Tempol, one of nitroxides, is a novel ultraviolet-A1 radiation protector for human dermal fibroblasts. J. Dermatol. Sci. 2005, 37:137-143.
60. Zhong H., Simons J.W. Direct comparison of GAPDH, beta-actin, cyclophilin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia. Biochem. Biophys. Res. Commun. 1999, 259:523-526.