Skip to main content
SpringerLink
Log in

Automated Fluorescent Differential Display for Cancer Gene Profiling

  • Protocol
  • First Online:
Cancer Gene Profiling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 576))

Summary

Since its invention in 1992, differential display (DD) has become the most commonly used technique for identifying differentially expressed genes because of its many advantages over competing technologies such as DNA microarray, serial analysis of gene expression (SAGE), and subtractive hybridization. A large number of these publications have been in the field of cancer, specifically on p53 target genes. Despite the great impact of the method on biomedical research, there had been a lack of automation of DD technology to increase its throughput and accuracy for systematic gene expression analysis. Many previous DD work has taken a “shotgun” approach of identifying one gene at a time, with a limited number of polymerase chain reactions (PCRs) set up manually, giving DD a low-tech and low-throughput image. We have optimized the DD process with a platform that incorporates fluorescent digital readout, automated liquid handling, and large-format gels capable of running entire 96-well plates. The resulting streamlined fluorescent DD (FDD) technology offers an unprecedented accuracy, sensitivity, and throughput in comprehensive and quantitative analysis of gene expression. These major improvements will allow researchers to find differentially expressed genes of interest, both known and novel, quickly and easily.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Sager, R. (1997) Expression genetics in cancer: shifting the focus from DNA to RNA. Proc. Natl. Acad. Sci. USA 94,952–955.

    Article  PubMed  CAS  Google Scholar 

  2. Vogelstein, B., Lane, D., and Levine, A.J. (2000) Surfing the p53 network. Nature 408, 307–310.

    Article  PubMed  CAS  Google Scholar 

  3. Liang, P., and Pardee, A.B. (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967–971.

    Article  PubMed  CAS  Google Scholar 

  4. Liang, P. (2002) A decade of differential display. Biotechniques 33, 338–346.

    PubMed  CAS  Google Scholar 

  5. Liang, P., and Pardee, A.B. (2003) Analysing differential gene expression in cancer. Nat. Rev. Cancer 3, 869–876.

    Article  PubMed  CAS  Google Scholar 

  6. Liang, P., Meade, J., and Pardee, A.B. (2007) A protocol for differential display of mRNA expression using either fluorescent or radioactive labeling. Nat. Protoc.2, 457–470.

    Article  PubMed  CAS  Google Scholar 

  7. 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 

  8. Chee, M., Yang, R., Hubbell, E., Berno, A., Huang, X.C., Stern, D., et al. (1996) Accessing genetic information with high-density DNA arrays. Science274, 610–614.

    Article  PubMed  CAS  Google Scholar 

  9. Velculescu V.E., Zhang L., Vogelstein B., and Kinzler K.W. (1995) Serial analysis of gene expression. Science 270, 484–487.

    Article  PubMed  CAS  Google Scholar 

  10. Zimmermann, C.R., Orr, W.C., Leclerc, R.F., Barnard, E.C., and Timberlake, W.E. (1980) Molecular cloning and selection of genes regulated in Aspergillus development. Cell21, 709–715.

    Article  PubMed  CAS  Google Scholar 

  11. McCarthy, S.A., Samuels, M.L., Pritchard, C.A., Abraham, J.A., and McMahon, M. (1995) Rapid induction of heparin-binding epidermal growth factor/diphtheria toxin receptor expression by Raf and Ras oncogenes. Genes Devel. 9, 1953–1964.

    Article  PubMed  CAS  Google Scholar 

  12. Zhang, R., Tan, Z., and Liang, P. (2000) Identification of a novel ligand-receptor pair constitutively activated by Ras oncogenes.J. Biol. Chem. 275, 24436–24443.

    Article  PubMed  CAS  Google Scholar 

  13. You, M., Ku, P.T., Hrdlickova, R., and Bose, H.R., Jr. (1997) ch-IAP1, a member of the inhibitor-of-apoptosis protein family, is a mediator of the antiapoptotic activity of the v-Rel oncoprotein. Mol. Cell. Biol. 17, 7328–7341.

    PubMed  CAS  Google Scholar 

  14. Park, B.-W., O’Rourke, D.M., Wang, Q., Davis, J.G., Post, A., Qian, X., et al. (1999) Induction of the Tat-binding protein 1 gene accompanies the disabling of oncogenic erbB receptor tyrosine kinases. Proc. Natl. Acad. Sci. USA 96, 6434–6438.

    Article  PubMed  CAS  Google Scholar 

  15. Wang, M., Tan, Z., Zhang, R., Kotenko, S.V., and Liang, P. (2002) Interleukin-24 (Mob-5/Mda-7) signals through two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2. J. Biol. Chem. 277, 7341–7347.

    Article  PubMed  CAS  Google Scholar 

  16. El-Deiry, W.S. (1998) Regulation of p53 downstream genes. Semin. Cancer Biol. 8, 345–357.

    Article  PubMed  CAS  Google Scholar 

  17. Wu, X., Bayle, J.H., Olson, D., and Levine, A.J. (1993) The p53-mdm-2 autoregulatory feedback loop. Genes Dev. 7, 1126–1132.

    Article  PubMed  CAS  Google Scholar 

  18. El-Deiry W.S., Tokino, T., Velculescu, V.E., Levy, D.B., Parsons, R., Trent, J.M., et al. (1993) WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817–825.

    Article  PubMed  CAS  Google Scholar 

  19. Miyashita, T., and Reed, J.C. (1995) Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80, 293–299

    Article  PubMed  CAS  Google Scholar 

  20. Okamoto, K., and Beach, D. (1994) Cyclin G is a transcriptional target of the p53 tumor suppressor protein. EMBO J. 13, 4816–4822.

    PubMed  CAS  Google Scholar 

  21. Buckbinder, L, Talbott, R., Velasco-Miguel, S., Takenaka, I., Faha, B., Seizinger, B.R., et al. (1995) Induction of the growth inhibitor IGF-binding protein 3 by p53. Nature 377, 646–649.

    Article  PubMed  CAS  Google Scholar 

  22. Polyak, K., Xia, Y., Zweier, J.L, Kinzler, K.W., and Vogelstein, B. (1997) A model for p53-induced apoptosis. Nature 389, 300–305.

    Article  PubMed  CAS  Google Scholar 

  23. Wu, G.S., Burns, T.F., McDonald, E.R., Jiang, W., Meng, R., Krantz, I.D., et al. (1997) KILLER/DR5 is a DNA damage-inducible p53-regulated death receptor gene. Nat. Genet. 17, 141–143.

    Article  PubMed  CAS  Google Scholar 

  24. Gu, Z., Flemington, C., Chittenden, T., and Zambetti, G.P. (2000) ei24, a p53 response gene involved in growth suppression and apoptosis. Mol. Cell. Biol. 20, 233–241.

    Article  PubMed  CAS  Google Scholar 

  25. Israeli, D., Tessler, E., Haupt, Y., Elkeles, A., Wilder, S., Amson, R., et al. (1997) A novel p53-inducible gene, PAG608, encodes a nuclear zinc finger protein whose overexpression promotes apoptosis. EMBO J. 16, 4384–4392.

    Article  PubMed  CAS  Google Scholar 

  26. Lo, P.K., Chen, J.-Y., Lo, W.-C., Chen, B.-F., Hsin, J.-P., Tang, P.-P, et al. (1999) Identification of a novel mouse p53 target gene DDA3. Oncogene 18, 7765–7774.

    Article  PubMed  CAS  Google Scholar 

  27. Takei, Y., Ishikawa, S., Tokino, T., Muto, T., and Nakamura, Y. (1998) Isolation of a novel TP53 target gene from a colon cancer cell line carrying a highly regulated wild-type TP53 expression system. Genes Chromosomes Cancer 23, 1–9.

    Article  PubMed  CAS  Google Scholar 

  28. Ng, C.C., Koyama, K., Okamura, S., Kondoh, H., Takei, Y., and Nakamura, Y. (1999) Isolation and characterization of a novel TP53-inducible gene, TP53TG3. Genes Chromosomes Cancer 26, 329–335.

    Article  PubMed  CAS  Google Scholar 

  29. Tanaka, H., Arakawa, H., Yamaguchi, T., Shiraishi, K., Fukuda, S., Matsui, K., et al. (2000) A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature 404, 42–49.

    Article  PubMed  CAS  Google Scholar 

  30. Attardi, L., Reczek, E.E., Cosmas, C., Demicco, E.G., McCurrach, M.E., Lowe, S.W., et al. (2000) PERP, an apoptosis-associated target of p53, is a novel member of the PMP-22/gas3 family. Genes Dev. 14, 704–718.

    PubMed  CAS  Google Scholar 

  31. Saller, E., Tom, E., Brunori, M., Otter, M., Estreicher, A., Mack, D.H., et al. (1999) Increased apoptosis induction by 121F mutant p53. EMBO J. 18, 4424–4437.

    Article  PubMed  CAS  Google Scholar 

  32. Oda, E., Ohki, R., Murasawa, H., Nemoto, J., Shibue, T., Yamashita, T., et al. (2000) Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 288, 1053–1058.

    Article  PubMed  CAS  Google Scholar 

  33. Lin, Y., Ma, W., and Benchimol, S. (2000) Pidd, a new death-domain-containing protein is induced by p53 and promotes apoptosis. Nat. Genet. 26, 124–127.

    Google Scholar 

  34. Oda, E., Arakawa, H., Tanaka, T., Matsuda, K., Tanikawa, C., Mori, T., et al. (2000) p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53. Cell 102, 849–862.

    Article  PubMed  CAS  Google Scholar 

  35. Okamura, S., Arakawa, H., Tanaka, T., Nakanishi, H., Ng, C.C., Taya, Y., et al. (2001) p53DINP1, a p53-inducible gene, regulates p53-dependent apoptosis. Mol. Cell 8, 85–94.

    Article  PubMed  CAS  Google Scholar 

  36. Yu, J., Zhang, L, Hwang, P.M., Kinzler, K.W., and Vogelstein, B. (2001) PUMA induces the rapid apoptosis of colorectal cancer cells. Mol. Cell 7, 673–682.

    Article  PubMed  CAS  Google Scholar 

  37. Nakano, K., and Vousden, K.H. (2001) PUMA, a novel proapoptotic gene, is induced by p53. Mol. Cell 7, 683–694.

    Article  PubMed  CAS  Google Scholar 

  38. Leng, R.P., Lin, Y., Ma, W., Wu, H., Lemmers, B., Chung, S., et al. (2003) Pirh2, a p53-induced ubiquitin-protein ligase, promotes p53 degradation. Cell 112, 779–791.

    Article  PubMed  CAS  Google Scholar 

  39. Yin, Y., Liu, Y.-X., Jin, Y.J., Hall, E.J., and Barrett, J.C. (2003) PAC1 phosphatase is a transcription target of p53 in signalling apoptosis and growth suppression. Nature 422, 527–531.

    Article  PubMed  CAS  Google Scholar 

  40. Owen-Schaub, L.B., Zhang, W., Cusack, J.C., Angelo, L.S., Santee, S.M., Fujiwara, T., et al. (1995) Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 expression. Mol. Cell. Biol. 15, 3032–3040.

    PubMed  CAS  Google Scholar 

  41. Kannan, K., Kaminski, N., Rechavi, G., Jakob-Hirsch, J., Amariglio, N., and Givol, D. (2001) DNA microarray analysis of genes involved in p53 mediated apoptosis: activation of Apaf-1. Oncogene 20, 3449–3455.

    Article  PubMed  CAS  Google Scholar 

  42. Stambolic, V., MacPherson, D., Sas, D., Lin, Y., Snow, B., Jang, Y., et al. (2001) Regulation of PTEN transcription by p53. Mol. Cell 8, 317–325.

    Article  PubMed  CAS  Google Scholar 

  43. Sax, J.K., Fei, P., Murphy, M.E., Bernhard, E., Korsmeyer, S.J., and El-Deiry, W.S. (2002) BID regulation by p53 contributes to chemosensitivity. Nat. Cell Biol. 411, 842–849.

    Article  CAS  Google Scholar 

  44. Cho, Y.-J., Meade, J.D., Walden, J.C., Chen, X., Guo, Z., and Liang, P. (2001) Multicolor fluorescent differential display. Biotechniques 30, 562–572.

    PubMed  CAS  Google Scholar 

  45. Meade, J.D., Cho, Y.-J., Fisher, J.S., Walden, J.C., Guo, Z., and Liang, P. (2005) Automation of fluorescent differential display with digital readout. In Differential Display Methods and Protocols, 2nd edition. Vol. 317 (Liang, P., Meade, J.D., & Pardee, A.B., eds.) Humana Press, Totowa, NJ, pp. 23–57.

    Google Scholar 

  46. Bauer, D., Muller, H., Reich, J., Riedel, H., Ahrenkiel, V., Warthoe, P., et al. (1993) Identification of differentially expressed mRNA species by an improved display technique (DDRT-PCR). Nucleic Acids Res. 21, 4272–4280.

    Article  PubMed  CAS  Google Scholar 

  47. Liang, P., Bauer, D., Averboukh, L., Warthoe, P., Rohrwild, M., Muller, H., et al. (1995) Analysis of altered gene expression by differential display. Methods Enzymol. 254, 304–321.

    Article  PubMed  CAS  Google Scholar 

  48. Liang, P., Zhu, W., Zhang, X., Guo, Z., O’Conell, R.P., Averboukh, L., et al. (1994) Differential Display using one-base anchored oligo-dT primers. Nucleic Acids Res. 22, 5763–5764.

    Article  PubMed  CAS  Google Scholar 

  49. Liang, P., Averboukh, L., and Pardee, A.B. (1994) Method of differential display. In Methods in Molecular Genetics, (Adolph, K.W., ed.) Academic, San Diego, CA, pp. 3–16.

    Google Scholar 

  50. Yang, S., and Liang, P. (2004) Global analysis of gene expression by differential display - a mathematical model. Mol. Biotechnol. 27, 197–208.

    Article  PubMed  CAS  Google Scholar 

  51. Liang, P., Averboukh, L., and Pardee, A.B. (1993) Distribution and cloning of eukaryotic mRNAs by means of differential display: Refinements and optimization. Nucleic Acids Res. 21, 3269–3275.

    Article  PubMed  CAS  Google Scholar 

  52. Hsu, D.K., Donohue, P.J., Alberts, G.F., and Winkles, J.A. (1993) Fibroblast growth factor-1 induces phosphofructokinase, fatty acid synthase and Ca (2+)-ATPase mRNA expression in NIH 3T3 cells. Biochem. Biophys. Res. Commun. 197, 1483–1491.

    Article  PubMed  CAS  Google Scholar 

  53. Sokolov, B.P., and Prockop, D.J. (1994) A rapid and simple PCR-based method for isolation of cDNAs from differentially expressed genes. Nucleic Acids Res. 22, 4009–4015.

    Article  PubMed  CAS  Google Scholar 

  54. Irie, T., Oshida, T., Hasegawa, H., Matsuoka, Y., Li, T., Oya, Y., et al. (2000) Automated DNA fragment collection by capillary array gel electrophoresis in search of differentially expressed genes. Electrophoresis 21, 367–374.

    Article  PubMed  CAS  Google Scholar 

  55. Ausubel, F., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., et al. (eds.) (1995) Short Protocols in Molecular Biology (3rd edition). Wiley, New York, NY. Section 4.9.1.–4.9.8.

    Google Scholar 

  56. Zhang, H., Zhang, R., and Liang, P. (1996) Differential screening of gene expression difference enriched by differential display. Nucleic Acids Res. 24, 2454–2455.

    Article  PubMed  CAS  Google Scholar 

  57. Ramdas, L., Coombes, K.R., Baggerly, K., Abruzzo, L., Highsmith, W.E., Krogmann, T., et al. (2001) Sources of nonlinearity in cDNA microarray expression measurements. Genome Biol. 2, RESEARCH0047.

    Article  PubMed  CAS  Google Scholar 

  58. Richmond, C.S., Glasner, J.D., Mau, R., Jin, H., and Blattner, F.R. (1999) Genome-wide expression profiling in Escherichia coli K-12. Nucleic Acids Res. 27, 3821–3835.

    Article  PubMed  CAS  Google Scholar 

  59. Gibbs, W.W. (2001) Shrinking to enormity: DNA microarrays are reshaping basic biology – but scientist fear that they may soon drown in data. Sci. Am. 284, 33–34.

    Article  CAS  Google Scholar 

  60. Liang, P. (2000) Gene discovery using differential display. Gen. Eng. News 20, 37.

    Google Scholar 

  61. Liang, S., Rossby, S.P., Liang, P., Shelton, R.C., Manier, D.H., Chakrabarti, A., et al. (2005) Detection of an mRNA polymorphism by differential display. In Differential Display Methods and Protocols, 2nd edition. Vol. 317 (Liang, P., Meade, J.D., & Pardee, A.B., eds.) Humana Press, Totowa, NJ, pp 279–285.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. GenHunter Corporation, Nashville, TN, USA

    Jonathan D. Meade, Blake R. Shester, Jamie C. Walden & Zhen Guo

  2. Department of Cell Biology, Vanderbilt-Ingram Cancer Center, School of Medicine, Vanderbilt University, Nashville, TN, USA

    Yong-jig Cho & Peng Liang

Authors
  1. Jonathan D. Meade
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yong-jig Cho
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Blake R. Shester
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jamie C. Walden
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhen Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Peng Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Medizinische Fak. Carl Gustav Carus, TU Dresden, Fetscherstr. 74, Dresden, 01307, Germany

    Robert Grützmann

  2. Klinik und Poliklinik für, TU Dresden, Fetscherstraße 74, Dresden, 01307, Germany

    Christian Pilarsky

Rights and permissions

Reprints and permissions

Copyright information

© 2009 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Meade, J.D., Cho, Yj., Shester, B.R., Walden, J.C., Guo, Z., Liang, P. (2009). Automated Fluorescent Differential Display for Cancer Gene Profiling. In: Grützmann, R., Pilarsky, C. (eds) Cancer Gene Profiling. Methods in Molecular Biology, vol 576. Humana Press. https://doi.org/10.1007/978-1-59745-545-9_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-545-9_7

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-934115-76-3

  • Online ISBN: 978-1-59745-545-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation