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T Cell Protocols pp 337–353Cite as

Transcriptional Expression Analysis of T-Cell Activation by Multiplex Messenger Assay (MMA)

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 134))

Abstract

Gene indices with many thousands of entries have been constructed by tag sequencing of randomly selected cDNA clones (1–7) and are widely available in repositories, such as the dbEST database (16). As more and more genes are identified, efforts are redirected towards understanding the control of gene expression that occurs in a strictly ordered time and cell-dependent fashion. Expression measurements often constitute a first step in this direction, and can be performed on a reasonably large scale using highly parallel hybridization methods. Hybridization methods, using complex probes and large arrays of targets, derive their power from the fact that each individual experiment provides a very large amount of information. Unrivaled for large-scale measurement of gene expression, these methods are based on the hybridization of complex probes with high-density colony cDNA (or PCR product) filters followed by quantitative measurement of the amount of hybridized probe on each colony. There are three key elements indispensable for accomplishing this task: the robot (for spotting colonies), the phosphoimager (for analyzing exposure), and the image analysis software (Fig. 1).

Technical approach: (Upper left) A robot to produce filters from 96- or 384-well plates. (Upper right) a filter hybrized with a vector probe showing the spotting patterns. The square A1 contains the clones at the position A1 from the 16 plates (96 wells). Spots localized at the bottom left of each square correspond to the control clone A. thaliana cytochrome c554 except for six spots toward the middle of the filter which contain the poly A control clones. Filters are exposed on a phosphor screen. (Lower right) the imaging plate device which transforms the hybridization signature into image files. (Lower left) the Bioimage software which quantifies intensity signals and assignes plate coordinates to each clone. Data are then transferred to EXCEL for further analysis.

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Notes

  1. 1.

    *

    Unfortunately this very useful resource is not completely error-free: Discrepancies in the correspondence between physical clones and tag sequences range from 10 to 20%, imposing expensive and time-consuming verification when these entities are used as reagents

References

  1. Adams, M. D., Kelley, J. M., Gocayne, J. D., Dubnick, M., Polymeropoulos, M. H., Xiao, H., Merril, C. R., Wu, A., Olde, B., Moreno, R. F., et al. (1991) Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252, 1651–1656.

    Article  PubMed  CAS  Google Scholar 

  2. Adams, M. D., Dubnick, M., Kerlavage, A. R., Moreno, R., Kelley, J. M., Utterback, T. R., Nagle, J. W., Fields, C., and Venter, J. C. (1992) Sequence identification of 2,375 human brain genes. Nature 355, 632–634.

    Article  PubMed  CAS  Google Scholar 

  3. Okubo, K., Hori, N., Matoba, R., Niiyama, T., Fukushima, A., Kojima, Y., and Matsubara, K. (1992) Large scale cDNA sequencing for analysis of quantitative and qualitative aspects of gene expression. Nat. Genet. 2, 173–179.

    Article  PubMed  CAS  Google Scholar 

  4. Adams, M. D., Soares, M. B., Kerlavage, A. R., Fields, C., and Venter, J. C. (1993) Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library. Nat. Genet. 4, 373–380.

    Article  PubMed  CAS  Google Scholar 

  5. Adams, M. D., Kerlavage, A. R., Fields, C., and Venter, J. C. (1993) Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence. Nat. Genet. 4, 256–267.

    Article  PubMed  CAS  Google Scholar 

  6. Takeda, J., Yano, H., Eng, S., Zeng, Y., and Bell, G. I. (1993) Construction of a normalized directionally cloned cDNA library from adult heart and analysis of 3040 clones by partial sequencing. Hum. Mol. Genet. 2, 1793–1798.

    Article  PubMed  CAS  Google Scholar 

  7. Sudo, K., Chinen, K., and Nakamura, Y. (1994) 2058 expressed sequence tags (ESTs) from a human fetal lung cDNA library. Genomics 24, 276–279.

    Article  PubMed  CAS  Google Scholar 

  8. Gress, T. M., Hoheisel, J. D., Lennon, G. G., Zehetner, G., and Lehrach, H. (1992) Hybridization fingerprinting of high-density cDNA-library arrays with cDNA pools derived from whole tissues. Mamm. Genome 3, 609–661.

    Article  PubMed  CAS  Google Scholar 

  9. Nguyen, C., Rocha, D., Granjeaud, S., Baldit, M., Bernard, K., Naquet, P., and Jordan, B. R. (1995) Differential gene expression in the murine thymus assayed by quantitative hybridization of arrayed cDNA clones. Genomics 29, 207–215.

    Article  PubMed  CAS  Google Scholar 

  10. 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 

  11. Zhao, N., Hashida, H., Takahashi, N., Misumi, Y., and Sakaki, Y. (1995) High-density cDNA filter analysis: a novel approach for large-scale, quantitative analysis of gene expression. Gene 156, 207–213.

    Article  PubMed  CAS  Google Scholar 

  12. Bernard, K., Auphan, N., Granjeaud, S., Victorero, G., Schmitt-Verhulst, A. M., Jordan, B. R., and Nguyen, C. (1996) Multiplex Messenger Assay: simultaneous, quantitative measurement of expression for many genes in the context of T cell activation. Nucleic Acids Res. 24, 1435–1443.

    Article  PubMed  CAS  Google Scholar 

  13. Pietu, G., Alibert, O., Guichard, V., Lamy, B., Bois, F., Leroy E., Mariage-Samson, R., Houlgatte, R., Soularue, P., and Auffray, C. (1996) Novel gene transcripts preferentially expressed in human muscles revealed by quantitative hybridization of a high density cDNA array. Genome Res. 6, 492–503.

    Article  PubMed  CAS  Google Scholar 

  14. Chee, M., Yang, R., Hubbell, E., Berno, A., Huang, X. C., Stern, D, Winkle, J., Lockhart, D. J., Morris, M. S., and Fodor, S. P. (1996) Accessing genetic information with high-density DNA arrays. Science 274, 610–614.

    Article  PubMed  CAS  Google Scholar 

  15. Rocha, D., Carrier, A., Naspetti, M., Victorero, G., Anderson, E., Botcherby, M., Nguyen, C., Naquet, P., and Jordan, B. R. (1997) Modulation of mRNA levels in the presence of thymocytes and genome mapping for a set of genes expressed in mouse thymic epithelial cells. Immunogenetics 46, 142–151.

    Article  PubMed  CAS  Google Scholar 

  16. .

    Google Scholar 

  17. Bertrand, R. Jordan (1998) Large scale expression measurement by hybridization methods: From high-density membranes to “DNA chips”. Jap. J. Biochem., in press.

    Google Scholar 

  18. 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 

  19. Schena, M., Shalon, D., Heller, R., Chai, A., Brown, P. O., and Davis, R. W. (1996) Parallel human genome analysis: microarray-based expression monitoring of 1000 genes. Proc. Natl. Acad. Sci. USA 93, 10,614–10,619.

    Article  PubMed  CAS  Google Scholar 

  20. Shalon, D., Smith, S. J., and Brown, P. O. A. (1996) DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization. Genome Res. 6(7), 639–645.

    Article  PubMed  CAS  Google Scholar 

  21. 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. Nature Gene. 14, 457–460.

    Article  CAS  Google Scholar 

  22. Derisi, J. L., Iyer, V. R., and Brown, P. O. (1997) Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278, 680–686.

    Article  PubMed  CAS  Google Scholar 

  23. Southern, E. M., Maskos, U., and Elder, J. K. (1992) Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models. Genomics 13, 1008–1017.

    Article  PubMed  CAS  Google Scholar 

  24. Lockhart, D. J., Dong, H., Byrne, M. C., Follettie, M. T., Gallo, M. V., Chee, M. S., Mittmann, M., Wang C., Kobayashi, M., Horton, H. and Brown, E. L. (1996) Expression monitoring by hybridization to high-density oligonucleotide arrays. Nature Biotechnol. 14, 1675–1680.

    Article  CAS  Google Scholar 

  25. IMAGE consortium

    Google Scholar 

  26. Clontech “ATLAS array”

    Google Scholar 

  27. Genome Systems “Gene discovery array:”.

    Google Scholar 

  28. Weiss, A. and Imboden, J. B. (1987) Cell surface molecules and early events involved in human T lymphocyte activation. Adv. Immunol. 41, 1–38.

    Article  PubMed  CAS  Google Scholar 

  29. Beltz, G. A., Jacobs, K. A., Eickbush, T. H., Cherbas, P. T., and Kafatos, F. C. (1983) Isolation of multigene families and determination of homologies by filter hybridization methods. Methods Enzymol. 100, 266–285.

    Article  PubMed  CAS  Google Scholar 

  30. Nizetic, D., Dramanac, R. and Lehrach, H. (1991). An improved bacterial colony lysis procedure enables direct DNA hybridization using short (10, 11 bases) oligonucleotides to cosmids. Nucleic Acids Res. 19, 182.

    Article  PubMed  CAS  Google Scholar 

  31. Sambrook, J., Fritsch, E. F., and Maniatis T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, book 1, pp. 7.3–7.5.

    Google Scholar 

  32. Gress, T. M., Müller-Pillasch, F., Adler, G., Zehetner, G., and Lehrach, H. (1984) European pancreatic cancer reference library system, EPCRLS. Eur. J. Cancer 30A, 1391–1394.

    Google Scholar 

  33. Johnston, R. F., Pickett, S. C. and Barker, D. L. (1990) Autroradiography using storage phosphor technology. Electrophoresis 11, 355–360.

    Article  PubMed  CAS  Google Scholar 

  34. Mory, K. and Hmaoka, T. (1994) IP autoradiographiy system (BAS). Protein Nucleic Acid Enzyme 39, 181–191.

    Google Scholar 

  35. Patterson, S. D. and Latte, G. I. (1993) Evaluation of storage phosphor imaging plating for quantitative analysis of 2-D gels using the Quest II system. Biotechniques 15, 1076–1083.

    PubMed  CAS  Google Scholar 

  36. Granjeaud, S., Nguyen, C., Rocha, D., Luton, R., and Jordan, B. R. (1996) From hybridization image to numerical values: a practical, high throughput quantification system for high density filter hybridizations. Gene. Anal. Biomol. Engineer. 12, 151–162.

    Article  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Genome Structure and Immune Function Lab Centre d’Immunologie, INSERM CNRS Marseille-Luminy, Marseille, France

    Karine Bernard, Samuel Granjeaud, Geneviève Victoréro & Catherine Nguyen

Authors
  1. Karine Bernard
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  2. Samuel Granjeaud
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  3. Geneviève Victoréro
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  4. Catherine Nguyen
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Editor information

Editors and Affiliations

  1. Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC

    Kelly P. Kearse

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© 2000 Humana Press Inc., Totowa, NJ

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Bernard, K., Granjeaud, S., Victoréro, G., Nguyen, C. (2000). Transcriptional Expression Analysis of T-Cell Activation by Multiplex Messenger Assay (MMA). In: Kearse, K.P. (eds) T Cell Protocols. Methods in Molecular Biology™, vol 134. Humana Press. https://doi.org/10.1385/1-59259-682-7:337

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  • DOI: https://doi.org/10.1385/1-59259-682-7:337

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-810-3

  • Online ISBN: 978-1-59259-682-9

  • eBook Packages: Springer Protocols

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