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Comprehensive Transcriptional Profiling of Human Epidermis, Reconstituted Epidermal Equivalents, and Cultured Keratinocytes Using DNA Microarray Chips

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Epidermal Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 585))

Abstract

Because of its accessibility, skin has been among the first organs analyzed using DNA microarrays; psoriasis, melanomas, carcinomas, chronic wound biopsies, and epidermal keratinocytes in culture have been intensely investigated. Skin has everything: stem cells, differentiation, signaling, inflammation, diseases, cancer, etc. Here we provide step-by-step instructions for bioinformatics analysis of transcriptional profiling of skin. Specifically, we describe the use of GCOS and RMA programs for initial normalization and selection of differentially expressed genes, DAVID and LOLA programs for annotation of genes, and statistically relevant identification of over- and under-represented functional and biological categories in identified gene sets, L2L and Venn diagrams for comparing multiple lists of genes, and oPOSSUM for identification of statistically over-represented transcription factor binding sites in the promoter regions of gene sets. The work can be a primer for researchers embarking on skinomics, the comprehensive analysis of transcriptional changes in the skin.

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References

  1. Quackenbush, J., Hegde, P., Qi, R., Abernathy, K., Gay, C., Dharap, S., Gaspard, R., Hughes, J. E., Snesrud, E., and Lee, N. (2003) Genomics. Microarrays–guilt by association. A concise guide to cDNA microarray analysis. Science 302, 240–241.

    Article  PubMed  CAS  Google Scholar 

  2. Lee, M. L., Kuo, F. C., Whitmore, G. A., and Sklar, J. (2000) Importance of replication in microarray gene expression studies: statistical methods and evidence from repetitive cDNA hybridizations. Proc Natl Acad Sci USA 97, 9834–9839.

    Article  PubMed  CAS  Google Scholar 

  3. Schena, M., Shalon, D., Davis, R. W., and Brown, P. O. (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470.

    Article  PubMed  CAS  Google Scholar 

  4. Bingham, J. L., Carrigan, P. E., Miller, L. J., and Srinivasan, S. (2008) Extent and diversity of human alternative splicing established by complementary database annotation and microarray analysis. Omics 12, 83–92.

    Article  PubMed  CAS  Google Scholar 

  5. Birney, E., Andrews, D., Bevan, P., Caccamo, M., Cameron, G., Chen, Y., Clarke, L., Coates, G., Cox, T., Cuff, J., Curwen, V., Cutts, T., Down, T., Durbin, R., Eyras, E., Fernandez-Suarez, X. M., Gane, P., Gibbins, B., Gilbert, J., Hammond, M., Hotz, H., Iyer, V., Kahari, A., Jekosch, K., Kasprzyk, A., Keefe, D., Keenan, S., Lehvaslaiho, H., McVicker, G., Melsopp, C., Meidl, P., Mongin, E., Pettett, R., Potter, S., Proctor, G., Rae, M., Searle, S., Slater, G., Smedley, D., Smith, J., Spooner, W., Stabenau, A., Stalker, J., Storey, R., Ureta-Vidal, A., Woodwark, C., Clamp, M., and Hubbard, T. (2004) Ensembl 2004. Nucleic Acids Res 32, D468–D470.

    Article  PubMed  CAS  Google Scholar 

  6. Iyer, V. R., Eisen, M. B., Ross, D. T., Schuler, G., Moore, T., Lee, J. C., Trent, J. M., Staudt, L. M., Hudson, J., Jr., Boguski, M. S., Lashkari, D., Shalon, D., Botstein, D., and Brown, P. O. (1999) The transcriptional program in the response of human fibroblasts to serum. Science 283, 83–87.

    Article  PubMed  CAS  Google Scholar 

  7. Wong, R., Tran, V., Morhenn, V., Hung, S. P., Andersen, B., Ito, E., Wesley Hatfield, G., and Benson, N. R. (2004) Use of RT-PCR and DNA microarrays to characterize RNA recovered by non-invasive tape harvesting of normal and inflamed skin. J Invest Dermatol 123, 159–167.

    Article  PubMed  CAS  Google Scholar 

  8. Banno, T., Gazel, A., and Blumenberg, M. (2004) The use of DNA microarrays in dermatology research. Retinoids 20, 1–4.

    Google Scholar 

  9. Blumenberg, M. (2006) DNA microarrays in dermatology and skin biology. Omics 10, 243–260.

    Article  PubMed  CAS  Google Scholar 

  10. Brem, H., Stojadinovic, O., Diegelmann, R. F., Entero, H., Lee, B., Pastar, I., Golinko, M., Rosenberg, H., and Tomic-Canic, M. (2007) Molecular markers in patients with chronic wounds to guide surgical debridement. Mol Med 13, 30–39.

    Article  PubMed  Google Scholar 

  11. Charles, C. A., Tomic-Canic, M., Vincek, V., Nassiri, M., Stojadinovic, O., Eaglstein, W. H., and Kirsner, R. S. (2008) A gene signature of nonhealing venous ulcers: potential diagnostic markers. J Am Acad Dermatol 19, 19.

    Google Scholar 

  12. Harsha, A., Stojadinovic, O., Brem, H., Sehara-Fujisawa, A., Wewer, U., Loomis, C. A., Blobel, C. P., and Tomic-Canic, M. (2008) ADAM12: a potential target for the treatment of chronic wounds. J Mol Med 86, 961–969.

    Article  PubMed  CAS  Google Scholar 

  13. Stojadinovic, O., Pastar, I., Vukelic, S., Mahoney, M. G., Brennan, D., Krzyzanowska, A., Golinko, M., Brem, H., and Tomic-Canic, M. (2008) Deregulation of keratinocyte differentiation and activation: a hallmark of venous ulcers. J Cell Mol Med 28, 28.

    Google Scholar 

  14. DeRisi, J., Penland, L., Brown, P. O., Bittner, M. L., Meltzer, P. S., Ray, M., Chen, Y., Su, Y. A., and Trent, J. M. (1996) Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nat Genet 14, 457–460.

    Article  PubMed  CAS  Google Scholar 

  15. Li, D., Turi, T. G., Schuck, A., Freedberg, I. M., Khitrov, G., and Blumenberg, M. (2001) Rays and arrays: the transcriptional program in the response of human epidermal keratinocytes to UVB illumination. FASEB J 15, 2533–2535.

    PubMed  CAS  Google Scholar 

  16. Sesto, A., Navarro, M., Burslem, F., and Jorcano, J. L. (2002) Analysis of the ultraviolet B response in primary human keratinocytes using oligonucleotide microarrays. Proc Natl Acad Sci USA 99, 2965–2970.

    Article  PubMed  CAS  Google Scholar 

  17. Murakami, T., Fujimoto, M., Ohtsuki, M., and Nakagawa, H. (2001) Expression profiling of cancer-related genes in human keratinocytes following non-lethal ultraviolet B irradiation. J Dermatol Sci 27, 121–129.

    Article  PubMed  CAS  Google Scholar 

  18. Takao, J., Ariizumi, K., Dougherty, II, and Cruz, P. D., Jr. (2002) Genomic scale analysis of the human keratinocyte response to broad-band ultraviolet-B irradiation. Photodermatol Photoimmunol Photomed 18, 5–13.

    Article  PubMed  CAS  Google Scholar 

  19. Howell, B. G., Wang, B., Freed, I., Mamelak, A. J., Watanabe, H., and Sauder, D. N. (2004) Microarray analysis of UVB-regulated genes in keratinocytes: downregulation of angiogenesis inhibitor thrombospondin-1. J Dermatol Sci 34, 185–194.

    Article  PubMed  CAS  Google Scholar 

  20. Curto, E. V., Lambert, G. W., Davis, R. L., Wilborn, T. W., and Dooley, T. P. (2002) Biomarkers of human skin cells identified using DermArray DNA arrays and new bioinformatics methods. Biochem Biophys Res Commun 291, 1052–1064.

    Article  PubMed  CAS  Google Scholar 

  21. Gazel, A., Ramphal, P., Rosdy, M., De Wever, B., Tornier, C., Hosein, N., Lee, B., Tomic-Canic, M., and Blumenberg, M. (2003) Transcriptional profiling of epidermal keratinocytes: comparison of genes expressed in skin, cultured keratinocytes, and reconstituted epidermis, using large DNA microarrays. J Invest Dermatol 121, 1459–1468.

    Article  PubMed  CAS  Google Scholar 

  22. Bonnet-Duquennoy, M., Abaibou, H., Tailhardat, M., Lazou, K., Bosset, S., Le Varlet, B., Cleuziat, P., and Kurfurst, R. (2006) Study of housekeeping gene expression in human keratinocytes using OLISA, a long-oligonucleotide microarray and q RT-PCR. Eur J Dermatol 16, 136–140.

    PubMed  CAS  Google Scholar 

  23. Kunz, M., Ibrahim, S. M., Koczan, D., Scheid, S., Thiesen, H. J., and Gross, G. (2004) DNA microarray technology and its applications in dermatology. Exp Dermatol 13, 593–606.

    Article  PubMed  CAS  Google Scholar 

  24. Sellheyer, K., and Belbin, T. J. (2004) DNA microarrays: from structural genomics to functional genomics. The applications of gene chips in dermatology and dermatopathology. J Am Acad Dermatol 51, 681–692; quiz 693–686.

    Article  PubMed  Google Scholar 

  25. Blumenberg, M. (2005) Skinomics. J Invest Dermatol 124, viii–x.

    Article  PubMed  CAS  Google Scholar 

  26. Della Vedova, G., and Dondi, R. (2003) A library of efficient bioinformatics algorithms. Appl Bioinformatics 2, 117–121.

    PubMed  Google Scholar 

  27. Banno, T., Gazel, A., and Blumenberg, M. (2004) Effects of tumor necrosis factor-alpha (TNF alpha) in epidermal keratinocytes revealed using global transcriptional profiling. J Biol Chem 279, 32633–32642.

    Article  PubMed  CAS  Google Scholar 

  28. Banno, T., Gazel, A., and Blumenberg, M. (2005) Pathway-specific profiling identifies the NF-{kappa}B-dependent tumor necrosis factor {alpha}-regulated genes in epidermal keratinocytes. J Biol Chem 280, 18973–18980.

    Article  PubMed  CAS  Google Scholar 

  29. Gazel, A., Banno, T., Walsh, R., and Blumenberg, M. (2006) Inhibition of JNK promotes differentiation of epidermal keratinocytes. J Biol Chem 281, 20530–20541.

    Article  PubMed  CAS  Google Scholar 

  30. Radoja, N., Gazel, A., Banno, T., Yano, S., and Blumenberg, M. (2006) Transcriptional profiling of epidermal differentiation. Physiol Genomics 5, 5.

    Google Scholar 

  31. Stojadinovic, O., Lee, B., Vouthounis, C., Vukelic, S., Pastar, I., Blumenberg, M., Brem, H., and Tomic-Canic, M. (2007) Novel genomic effects of glucocorticoids in epidermal keratinocytes: inhibition of apoptosis, interferon-gamma pathway, and wound healing along with promotion of terminal differentiation. J Biol Chem 282, 4021–4034.

    Article  PubMed  CAS  Google Scholar 

  32. Rheinwald, J. G., and Green, H. (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331–344.

    Article  PubMed  CAS  Google Scholar 

  33. Randolph, R. K., and Simon, M. (1994) Characterization of retinol metabolism in cultured human epidermal keratinocytes. J Biol Chem 268, 9198–9205.

    Google Scholar 

  34. Bernard, F. X., Pedretti, N., Rosdy, M., and Deguercy, A. (2002) Comparison of gene expression profiles in human keratinocyte mono-layer cultures, reconstituted epidermis and normal human skin; transcriptional effects of retinoid treatments in reconstituted human epidermis. Exp Dermatol 11, 59–74.

    Article  PubMed  CAS  Google Scholar 

  35. Rosdy, M., and Clauss, L. C. (1990) Terminal epidermal differentiation of human keratinocytes grown in chemically defined medium on inert filter substrates at the air-liquid interface. J Invest Dermatol 95, 409–414.

    Article  PubMed  CAS  Google Scholar 

  36. Mahadevappa, M., and Warrington, J. A. (1999) A high-density probe array sample preparation method using 10- to 100-fold fewer cells. Nat Biotechnol 17, 1134–1136.

    Article  PubMed  CAS  Google Scholar 

  37. Li, D., Turi, T. G., Schuck, A., Freedberg, I. M., Khitrov, G., and Blumenberg, M. (2001) Rays and arrays: the transcriptional program in the response of human epidermal keratinocytes to UVB illumination. FASEB J 15, 2533–2535.

    PubMed  CAS  Google Scholar 

  38. Gazel, A., Ramphal, P., Rosdy, M., De Wever, B., Tornier, C., Hosein, N., Lee, B., Tomic-Canic, M., and Blumenberg, M. (2003) Transcriptional profiling of epidermal keratinocytes: comparison of genes expressed in skin, cultured keratinocytes, and reconstituted epidermis, using large DNA microarrays. J Invest Dermatol 121, 1459–1468.

    Article  PubMed  CAS  Google Scholar 

  39. Banno, T., Adachi, M., Mukkamala, L., and Blumenberg, M. (2003) Unique keratinocyte-specific effects of interferon-gamma that protect skin from viruses, identified using transcriptional profiling. Antivir Ther 8, 541–554.

    PubMed  CAS  Google Scholar 

  40. Yano, S., Banno, T., Walsh, R., and Blumenberg, M. (2008) Transcriptional responses of human epidermal keratinocytes to cytokine IL-1. J Cell Physiol 214, 1–13.

    Article  PubMed  CAS  Google Scholar 

  41. Bolstad, B. M., Irizarry, R. A., Astrand, M., and Speed, T. P. (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19, 185–193.

    Article  PubMed  CAS  Google Scholar 

  42. Rouillard, J. M., Herbert, C. J., and Zuker, M. (2002) OligoArray: genome-scale oligonucleotide design for microarrays. Bioinformatics 18, 486–487.

    Article  PubMed  CAS  Google Scholar 

  43. Eisen, M. B., Spellman, P. T., Brown, P. O., and Botstein, D. (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95, 14863–14868.

    Article  PubMed  CAS  Google Scholar 

  44. Eisen, M. B., Spellman, P. T., Brown, P. O., and Botstein, D. (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95, 14863–14868.

    Article  PubMed  CAS  Google Scholar 

  45. Ho Sui, S. J., Mortimer, J. R., Arenillas, D. J., Brumm, J., Walsh, C. J., Kennedy, B. P., and Wasserman, W. W. (2005) oPOSSUM: identification of over-represented transcription factor binding sites in co-expressed genes. Nucleic Acids Res 33, 3154–3164.

    Article  PubMed  Google Scholar 

  46. Gazel, A., Nijhawan, R. I., Walsh, R., and Blumenberg, M. (2008) Transcriptional profiling defines the roles of ERK and p38 kinases in epidermal keratinocytes. J Cell Physiol 215, 292–308.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. The Departments of Dermatology and Biochemistry, The NYU Cancer Institute, New York University School of Medicine, New York, NY, USA

    Ding-Dar Lee (Faculty of Medicine) & Miroslav Blumenberg

  2. Department of Dermatology, Taipei Veterans General Hospital, National Yang-Ming University, Taipei, Taiwan

    Ding-Dar Lee (Faculty of Medicine)

  3. Department of Pathology and NYU Cancer Institute, New York University School of Medicine, New York, NY, USA

    Jiri Zavadil

  4. Hospital for Special Surgery, Tissue Repair Lab, Tissue Engineering, Regeneration and Repair Program, and Department of Dermatology, Weill Medical College of the Cornell University, New York, NY, USA

    Marjana Tomic-Canic

Authors
  1. Ding-Dar Lee
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  2. Jiri Zavadil
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  3. Marjana Tomic-Canic
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  4. Miroslav Blumenberg
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Editor information

Editors and Affiliations

  1. Sprott Centre for Stem Cell Research, Ottawa Hosp. Research Institute (OHRI), Smyth Road 501, Ottawa, K1H 8L6, Canada

    Kursad Turksen

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© 2010 Humana Press, a part of Springer Science+Business Media, LLC

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Lee, DD., Zavadil, J., Tomic-Canic, M., Blumenberg, M. (2010). Comprehensive Transcriptional Profiling of Human Epidermis, Reconstituted Epidermal Equivalents, and Cultured Keratinocytes Using DNA Microarray Chips. In: Turksen, K. (eds) Epidermal Cells. Methods in Molecular Biology, vol 585. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-380-0_15

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  • DOI: https://doi.org/10.1007/978-1-60761-380-0_15

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-379-4

  • Online ISBN: 978-1-60761-380-0

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