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Arthritis Research pp 329–347Cite as

Differential Display Reverse Transcription-Polymerase Chain Reaction to Identify Novel Biomolecules in Arthritis Research

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Part of the book series: Methods in Molecular Medicine ((MIMM,volume 136))

Abstract

Differential display is one of the simplest techniques for discovering novel transcripts when comparing gene expression in biological systems. The method can be carried out on small amounts of total RNA and permits the simultaneous comparison of multiple independent samples in a single experiment. The methodology is versatile in that it allows the researcher to adapt the existing protocol by varying the selection of oligonucleotide primers used in the PCR. The putative differentials, which are isolated from the polyacrylamide gels as cDNA fragments of approx 100 to 500 bases in size, can be instantly recognized after sequencing by searching the nucleotide databases. However, one of the drawbacks is the isolation of false positives and hence the need to confirm the results of the screen by another method. Once the true differentials have been identified, further downstream work is also required to recognize which splice variant of the transcript gives rise to the expression differences and whether the gene expression results are corroborated at the protein level.

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References

  1. 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  CAS  PubMed  Google Scholar 

  2. Welsh, J., Chada, K., Dalal, S. S., Cheng, R., Ralph, D., and McClelland, M. (1992) Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res. 20, 4965–4970.

    Article  CAS  PubMed  Google Scholar 

  3. Van Der Pouw Kraan, T. C. T. M., Van Gaalen, F. A., Kasperkoitz, P. V., et al. (2003) Rheumatoid arthritis is a heterogeneous disease. Evidence for differences in the activation of the STAT-1 pathway between rheumatoid tissues. Arthritis Rheum. 48, 2132–2145.

    Article  PubMed  Google Scholar 

  4. Aidinis, V., Plows, D., Haralambous, S., et al. (2003) Functional analysis of an arthritogenic synovial fibroblast. Arthritis Res. Ther. 5, R140–R157.

    Article  CAS  PubMed  Google Scholar 

  5. Olsen, N., Sokka, T., Seehorn, C. L., Kraft, B., Maas, K., Moore, J., and Aune, T.M. (2004) A gene expression signature for recent onset rheumatoid arthritis in peripheral blood mononuclear cells. Ann. Rheum. Dis. 63, 1387–1392.

    Article  CAS  PubMed  Google Scholar 

  6. Callard, D., Lescure, B., and Mazzolini, L. (1994) A method for the elimination of false positives generated by the mRNA differential display technique. BioTechniques 16, 1096–1103.

    CAS  PubMed  Google Scholar 

  7. Debouck, C. (1995) Differential display or differential dismay? Curr. Opin. Biotech. 6, 597–599.

    Article  CAS  Google Scholar 

  8. Ali, M., Ponchel, F., Wilson, K. E., et al. (2001) Rheumatoid arthritis synovial T cells regulate transcription of several genes associated with antigen-induced anergy. J. Clin. Invest. 107 519–528.

    Article  CAS  PubMed  Google Scholar 

  9. Mangasser-Stephan, K., Dooley, S., Welter, C., Mutschler, W., and Hanselmann, R.G. (1997) Identification of human semaphorin E gene expression in rheumatoid synovial cells by mRNA differential display. Biochem. Biophys. Res. Commun. 234, 153–156.

    Article  CAS  PubMed  Google Scholar 

  10. Kullmann, F., Judex, M., Ballhorn, W., et al. (1999) Kinesin-like protein CENPE is upregulated in rheumatoid synovial fibroblasts. Arthritis Res. 1, 71–80.

    Article  CAS  PubMed  Google Scholar 

  11. Hu, S.-I., Carozza, M., Klein, M., Nantermet, P., Luk, D., and Crowl, R. M. (1998) Human HtrA, an evolutionary conserved serine protease identified as a differentially expressed gene product in osteoarthritic cartilage. J. Biol. Chem. 273, 34406–34412.

    Article  CAS  PubMed  Google Scholar 

  12. Patel, I. R., Attur, M. G., Patel, R. N., et al. (1998) TNF-α convertase enzyme from human arthritis-affected cartilage: isolation of cDNA by differential display, expression of the active enzyme, and regulation of TNF-α. J. Immunol. 160, 4570–4579

    CAS  PubMed  Google Scholar 

  13. Attur, M. G., Dave, M. N., Stuchin, S., et al. (2001) Osteopontin. An intrinsic inhibitor of inflammation in cartilage. Arthritis Rheum. 44, 578–584.

    CAS  Google Scholar 

  14. Yamada, E., Ishiguro, N., Miyaishi, O., et al. (1997) Differential display analysis of murine collagen-induced arthritis: cloning of the cDNA-encoding murine ATPase inhibitor. Immunology 92, 571–576.

    Article  CAS  PubMed  Google Scholar 

  15. Sakurai, D., Yamaguchi, A., Tsuchiya, N., Yamamoto, K., and Tokunaga, K. (2001) Expression of ID family genes in the synovia from patients with rheumatoid arthritis. Biochem. Biophys. Res. Commun. 284, 436–442.

    Article  CAS  PubMed  Google Scholar 

  16. Judex, M., Neumann, E., Lechner, S., et al. (2003) Laser-mediated microdissection facilitates analysis of area-specific gene expression in rheumatoid synovium. Arthritis Rheum. 48, 97–102.

    Article  CAS  PubMed  Google Scholar 

  17. Ali, M., Markham, A.F., and Isaacs, J.D. (2001) Application of differential display to immunological research. J. Immunol. Methods 250, 29–43.

    Article  CAS  PubMed  Google Scholar 

  18. 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  CAS  PubMed  Google Scholar 

  19. Mathieu-Daude, F., Cheng, R., Welsh, J., and McClelland, M. (1996) Screening of differentially amplified cDNA products from RNA arbitrarily primed PCR fingerprints using single strand conformation polymorphism (SSCP) gels. Nucleic Acids Res. 24, 1504–1507.

    Article  CAS  PubMed  Google Scholar 

  20. Smith, N. R., Li, A., Aldersley, M., et al. (1997) Rapid determination of the complexity of cDNA bands extracted from DDRT-PCR polyacrylamide gels. Nucleic Acids Res. 25, 3553–3554.

    Article  Google Scholar 

  21. Martin, K. J., Kwan, C. P., O’Hare, M. J., Pardee, A.B., and Sager, R. (1998) Identification and verification of differential display cDNAs using gene-specific primers and hybridization arrays. BioTechniques 24, 1018–1026.

    CAS  PubMed  Google Scholar 

  22. Schmittgen, T. D. and Zakrajsek, B. A. (2000) Effect of experimental treatment on housekeeping gene expression validated by real-time, quantitative RT-PCR. J. Biochem. Biophys. Methods 46, 69–81.

    Article  CAS  PubMed  Google Scholar 

  23. Donohue, P. J., Alberts, G. F., Guo, Y., and Winkles, J.A. (1995) Identification by targeted differential display of an immediate early gene encoding a putative serine/threonine kinase. J. Biol. Chem. 270, 10351–10357.

    Article  CAS  PubMed  Google Scholar 

  24. Chuaqui, R. F., Englert, C. R., Strup, S. E., et al. (1997) Identification of a novel transcript up-regulated in a clinically aggressive prostate carcinoma. Urology 50, 302–307.

    Article  CAS  PubMed  Google Scholar 

  25. Ryoo, H. M., Hoffmann, H. M., Beumer, T., et al. (1997) Stage-specific expression of Dlx-5 during osteoblast differentiation: involvement in regulation of osteocalcin gene expression. Mol. Endocrinol. 11, 1681–1694.

    Article  CAS  PubMed  Google Scholar 

  26. Stone, B. and Warton, W. (1994) Targeted RNA fingerprinting: the cloning of differentially-expressed cDNA fragments enriched for members of the zinc finger gene family. Nucleic Acids Res. 22, 2612–2618.

    Article  CAS  PubMed  Google Scholar 

  27. Tohonen, V., Osterlund, C., and Nordqvist, K. (1998) Testatin: a cystatin-related gene expressed during early testis development. Proc. Natl. Acad. Sci. USA 95, 14208–14213.

    Article  CAS  PubMed  Google Scholar 

  28. Yoshikawa, T., Xing, G. Q., and Detera-Wadleigh, S.D. (1995) Detection, simultaneous display and direct sequencing of multiple nuclear hormone receptor genes using bilaterally targeted RNA fingerprinting. Biochim. Biophys. Acta 1264, 63–71.

    PubMed  Google Scholar 

  29. Liu, G., Takano, T., Matsuzuka, F., Higashiyama, T., Kuma, K., and Amino, N. (1999) Screening of specific changes in mRNAs in thyroid tumors by sequence specific differential display: decreased expression of c-fos mRNA in papillary carcinoma. Endocrine J. 46, 459–466.

    Article  CAS  Google Scholar 

  30. Clinton, M. and Scougall, R.K. (1995) Detection and capture of 35S-labeled gas released from reaction tubes during differential display PCR. BioTechniques 19, 798–799.

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  32. Dominguez, O., Ashhab, Y., Sabater, L., Belloso, E., Caro, P., and Pujol-Borrell, R. (1998) Cloning the ARE-containing genes by AU-motif-directed display. Genomics 54, 278–286.

    Article  CAS  PubMed  Google Scholar 

  33. Utans-Schneitz, U., Lorez, H., Klinkert, W. E., Da Silva, J., and Lesslauer W. (1998) A novel rat CC chemokine, identified by targeted differential display, is upregulated in brain inflammation. J. Neuroimmunol. 92, 179–190.

    Article  CAS  PubMed  Google Scholar 

  34. Guimaraes, J. M., Lee, F., Zlotnik, A., and McClanahan, T. (1995) Differential display by PCR: novel findings and applications. Nucleic Acids Res. 23, 1832–1833.

    Article  CAS  PubMed  Google Scholar 

  35. Sun, Y., Hegamyer, G., and Colburn, N.H. (1994) Molecular cloning of five messenger RNAs differentially expressed in preneoplastic or neoplastic JB6 mouse epidermal cells: one is homologous to human tissue inhibitor of metalloproteinases-3. Cancer Res. 54, 1139–1144.

    CAS  PubMed  Google Scholar 

  36. Rajeevan, M.S., Ranamukhaarachchi, D. G., Vernon, S.D., and Unger, E. R. (2001) Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies. Methods 25, 443–451.

    Article  CAS  PubMed  Google Scholar 

  37. Lee, Y. H., Tokraks, S., Pratley, R. E., Bogardus, C., and Permana, P. A. (2003) Identification of differentially expressed genes in skeletal muscle of non-diabetic insulin-resistant and insulin-sensitive Pima Indians by differential display PCR. Diabetologia 46, 1567–1575.

    Article  CAS  PubMed  Google Scholar 

  38. Toki, H., Namikawa, K., Su, Q., Kiryu-Seo, S., Sato, K., and Kiyama, H. (1998) Enhancement of extracellular glutamate scavenge system in injured motorneurons. J. Neurochem. 71, 913–919.

    Article  CAS  PubMed  Google Scholar 

  39. Bryant, Z., Subrahmanyan, L., Tworoger, M., et al. (1999) Characterization of differentially expressed genes in purified Drosophila follicle cells: toward a general strategy for cell type-specific developmental analysis. Proc. Natl. Acad. Sci. USA 96, 5559–5564.

    Article  CAS  PubMed  Google Scholar 

  40. Kim, J. W., Kim, S. J., Han, S. M., et al. (1998) Increased glyceraldehyde-3-phosphate dehydrogenase gene expression in human cervical cancers. Gynecol. Oncol. 71, 266–269.

    Article  CAS  PubMed  Google Scholar 

  41. Goidin, A., Mamessier, A., Staquet, M. J., Schmitt, D., and Berthier-Vergnes, O. (2001) Ribosomal 18SRNA prevails over glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and non-invasive human melanoma cell subpopulations. Anal. Biochem. 295, 17–21.

    Article  CAS  PubMed  Google Scholar 

  42. Hamalainen, H. K., Tubman, J. C., Vikman, S., et al. (2001) Identification and validation of endogenous reference genes for expression profiling of T helper cell differentiation by quantitative real-time PCR. Anal. Biochem. 229, 63–70.

    Article  Google Scholar 

  43. Glare, E. M., Divjak, M., Bailey, M. J., and Walters, E. H. (2002) β-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels. Thorax 57, 765–770.

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Leeds Institute of Molecular Medicine, University of Leeds, St. James’s University Hospital, Leeds, UK

    Manir Ali

  2. Institute of Cellular Medicine, University of Newcastle Upon Tyne, Newcastle Upon Tyne, UK

    John D. Isaacs

Authors
  1. Manir Ali
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  2. John D. Isaacs
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Editor information

Editors and Affiliations

  1. The Kennedy Institute of Rheumatology, Imperial College London, London, UK

    Andrew P. Cope

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© 2007 Humana Press Inc., Totowa, NJ

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Ali, M., Isaacs, J.D. (2007). Differential Display Reverse Transcription-Polymerase Chain Reaction to Identify Novel Biomolecules in Arthritis Research. In: Cope, A.P. (eds) Arthritis Research. Methods in Molecular Medicine, vol 136. Humana Press. https://doi.org/10.1007/978-1-59745-402-5_23

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  • DOI: https://doi.org/10.1007/978-1-59745-402-5_23

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-918-5

  • Online ISBN: 978-1-59745-402-5

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