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Approaches to the analysis of gene expression using mRNA

A technical overview

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Abstract

Messenger RNA is the blueprint for all proteins expressed within living systems. Therefore, the study of mRNA expression within normal and diseased tissues is central to our understanding of biological systems. However, blueprints, in themselves, perform no function unless they are used to produce the material for which they code. Spurious results may frequently result from poorly designed or inappropriate studies. This review seeks to highlight both the pitfalls and the promise of various approaches to the analysis of mRNA in different systems and to place these studies in the wider context of research approaches aimed at understanding the function of living systems. The various techniques for the analysis of mRNA are discussed, with particular reference to their potential uses and problems and relevant examples are cited from the literature. It is hoped that this overview of the uses of analytical approaches will allow both the novice researcher and the more experienced scientist to better structure research approaches.

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References

  1. Differential Display Methods & Protocols, in Methods in Molecular Biology, Vol. 85 (Liang, P. and Pardee, A. B., eds.), (1998) Humana Press, Totowa, NJ.

    Google Scholar 

  2. PRINS and In Situ PCR Protocols in Methods in Molecular Biology, Vol. 71 (Gosden J. R., ed), (1984) Humana Press, Totowa, NJ.

    Google Scholar 

  3. PCR Protocols, Current Methods & Applications, in Methods in Molecular Biology, Vol 15 (White, B. A., eds.), (1984) Humana Press, Totowa, NJ.

    Google Scholar 

  4. Ovarian Cancer: Methods & Protocols, in Methods in Molecular Medicine, Vol. 39 (Bartlett, J. M. S., ed.), (2000) Humana Press, Totowa, NJ.

    Google Scholar 

  5. US Dept Commerce Molecular Biology Techniques Forum, http://research.nwfsc.noaa.gov/protocols/methods/

  6. Miller, W. R., Hulme, M. J., Bartlett, J. M. S., MacCallum, J., and Dixon, J. M. (1997) Changes in messenger RNA expression of protein kinase A regulatory subunit ialpha in breast cancer patients treated with tamoxifen. Clin. Cancer Res. 3, 2399–2404.

    PubMed  CAS  Google Scholar 

  7. Bartlett, J. M., Langdon, S. P., Scott, W. N., et al. (1997) Transforming growth factor-beta isoform expression in human ovarian tumours. Eur. J. Cancer 33, 2397–2403.

    Article  PubMed  CAS  Google Scholar 

  8. Crickard, K., Gross, J. L., Crickard, U., et al. (1994) Basic fibroblast growth factor and receptor expression in human ovarian cancer. Gynecologic Oncol. 55, 277–284.

    Article  CAS  Google Scholar 

  9. Ji, H. J., Zhang, Q. Q., and Leung, B. S. (1990) Survey of oncogene and growth factor-receptor gene expression in cancer cells by intron-differential RNA/PCR. Biochem Biophys. Res. Commun. 170, 569–575.

    Article  PubMed  CAS  Google Scholar 

  10. Jindal, S. K., Ishii, E., Letarte, M., Vera, S., Teerds, K. J., and Dorrington, J. H. (1995) Regulation of transforming growth factor alpha gene expression in an ovarian surface epithelial cell line derived from a human carcinoma. Biol. Reprod. 52, 1027–1037.

    Article  PubMed  CAS  Google Scholar 

  11. Bandowski, J. J. (2000) Standardization of molecular amplification testing in the clinical microbiology laboratory. Clin. Microbiol. News 22, 185–190.

    Article  Google Scholar 

  12. Diachenko, L. B., Ledesma, J., Chenchik, A. A., and Siebert, P. D. (1996) Combining the technique of RNA fingerprinting and differential display to obtain differentially expressed messenger-RNA. Biochem. Biophys. Res. Commun. 219, 824–828.

    Article  PubMed  CAS  Google Scholar 

  13. Schneeberger, C., Eder, S., Speiser, P., and Zeillinger, R. (1996) Competitive reverse transcriptase-PCR: an improved method for determination of c-erbB-2 gene expression. Anticancer Res. 16, 849–852.

    PubMed  CAS  Google Scholar 

  14. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, New York.

    Google Scholar 

  15. Doyle, K., ed. (1996) Protocols and applications guide. Promega Corporation USA (Available free of charge).

  16. Brandt, B., Beckmann, A., Roetger, A. and Gebhardt, F. (2000) Double and competitive differential PCR for gene dosage quantitation. Chapter 38, pp. 347–356. In Ovarian Cancer: Methods & Protocols (Bartlett, J. M. S., ed). Vol. 39 Methods in Molecular Medicine. 2000 Human Press, Totowa New Jersey USA.

    Chapter  Google Scholar 

  17. Perez, R., Pendyala, L., Elakawi, Z., and Mahmoud Abu-hadid (2000) RT-PCR quantitation of HSP60 mRNA expression. Chapter 48, pp. 439–447. In: Ovarian Cancer: Methods & Protocols (Bartlett, J. M. S., ed.) Vol. 39 Methods in Molecular Medicine. Human Press, Totowa, New Jersey USA.

    Chapter  Google Scholar 

  18. Bartlett, J. M. S. (2000) RNAse protection assay analysis of mRNA for TGFβ1–3 in ovarian tumours: Chapter 47, pp. 431–437. In Ovarian Cancer: Methods & Protocols (Bartlett, J. M. S., ed.) Volume 39 Methods in Molecular Medicine. Humana Press, Totowa New Jersey USA.

    Chapter  Google Scholar 

  19. Sellar, G. C., Rabiasz, G. J., Smith, B. and Southgate, J. (2000) Differential Display. Chapter 50, pp 459–468. In: Ovarian Cancer: Methods & Protocols (Bartlett, J. M. S., ed.) Vol. 39 Methods in Molecular Medicine. Human Press, Totowam New Jersey USA.

    Chapter  Google Scholar 

  20. Bartlett, J. M. S., Hulme, J. J., and Miller, W. R. (1996) Analysis of cAMP RI-alpha messenger-RNA expression in breast-cancer-evaluation of quantitative polymerase chain-reaction for routine use. Brit. J. Cancer 73, 1538–1544.

    PubMed  CAS  Google Scholar 

  21. Underwood, M. A., Bartlett, J. M. S., and Cooke, T. G. (1994). An improved method for semiquantification of gene amplification from archival material. PCR Meth. Applic. 4, 178–184.

    CAS  Google Scholar 

  22. Peck, K., Sher, Y.-P., Shih, J.-Y., Roffler, S. R., Wu, C. W., and Yang, P.-C. (1998) Detection and quantitation of circulating cancer cells in the peripheral blood of lung cancer patients. Cancer Res. 58, 2761–2765.

    PubMed  CAS  Google Scholar 

  23. Ozawa, K., Seo, M., and Imamura, T., (1997) A quantitative method for evaluation of FGF family and FGF receptor family gene expression by RT-PCR. Brain Research Protocols 1, 211–216.

    Article  PubMed  CAS  Google Scholar 

  24. Larsson, M., Stahl, S., Uhlen, M., Wennborg, A. (2000) Expression profile viewer (ExProView): A software tool for transcriptome analysis. Genomics 63(3), 341–353.

    Article  PubMed  CAS  Google Scholar 

  25. Lash, A. E., Tolstoshev, C. M., Wagner, L., et al. (2000) SAGEmap: A public gene expression resource. Genome Research 10(7), 1051–1060.

    Article  PubMed  CAS  Google Scholar 

  26. http://www.sagenet.org.

  27. www.ncbi.nlm.nih.gov/sage.

  28. To, K.-Y. (2000) Identification of differential gene expression by high throughput analysis. Combinatorial Chemistry & High Throughput Screening 3(3), 235–241.

    CAS  Google Scholar 

  29. Carulli, J. P., Artinger, M., Swain, P. M., et al. (1998) High throughput analysis of differential gene expression. J. Cell. Biochem. Issue SUPPL. 30/31 (pp 286–296).

  30. Bartlett, J. M. S. Technology evaluation: SAGE. (2001) Curr. Opin. Molec. Therapeu. 3, 85–96.

    CAS  Google Scholar 

  31. Gygi, S. P., Rochon, Y., Franza, B. R., and Aebersold, R. (1999) Correlation between protein and mRNA abundance in yeast. Molecular & Cellular Biology 19(3), 1720–30.

    CAS  Google Scholar 

  32. Lockhart, D., Dong, H., Byrne, M., et al. (1996) Expression monitoring by hybridization to high-density oligonucleotide arrays. Nature Biotechnology 14, 1675–14,1680.

    Article  PubMed  CAS  Google Scholar 

  33. Schena, M., Shalon, D., Davis, R., and Brown, P. (1995) Quantitative monitoring of gene expression patterns with a complementary cDNA microarray. Science 270467–270470.

  34. Lomax, M. I., Huang, L., Cho, Y., Gong, T.-W., and Altschuler, R.A. (2000) Differential display and gene arrays to examine auditory plasticity. Hearing Research 147(1–2), 293–302.

    Article  PubMed  CAS  Google Scholar 

  35. Sun, Y., (2000) Identification and characterization of genes responsive to apoptosis: Application of DNA chip technology and mRNA differential display. Histology & Histopathology 15(4), 1271–1284.

    CAS  Google Scholar 

  36. Jurecic, R. and Belmont, J. W. (2000) Long-distance DD-PCR and cDNA microarrays. Current Opinion in Microbiology 3(3), 316–321.

    Article  PubMed  CAS  Google Scholar 

  37. http://www.genzyme.com/sage/temp.htm

  38. Marguiles, E. and Innis, J. (2000) eSAGE: Managing and analysing data generated with serial analysis of gene expression (SAGE). Bioinformatics 16(7), 650–651.

    Article  Google Scholar 

  39. Lian, P. and Pardee, A. B. (1998) Differential display: A general protocol. Molec. Biotech. 10(3), 261–267.

    Google Scholar 

  40. Jorgensen, M., Bevort, M., Kledal, T. S., Hansen, B. V., Dalgaard, M., and Leffers, H. (1999) Differential display competitive polymerase chain reaction: An optimal tool for assaying gene expression. Electrophoresis 20(2), 230–240.

    PubMed  CAS  Google Scholar 

  41. Matz, M. V. and Lukyanov, S. A. (1998) Different strategies of differential display: Areas of application. Nucleic Acids Research 26(24), 5537–5543.

    Article  PubMed  CAS  Google Scholar 

  42. Lapointe, J. and Labrie, C., (1999) Identification and cloning of a novel androgen-responsive gene, uridine diphosphoglucose dehydrogenase, in human breast cancer cells. Endocrinology 140(10), 4486–4493.

    Article  PubMed  CAS  Google Scholar 

  43. Cirelli, C., Tononi, G. (1999) Differences in brain gene expression between sleep and waking as revealed by mRNA differential display and cDNA microarray technology. J. Sleep Res. 8(1), 44–52.

    Article  PubMed  Google Scholar 

  44. Aittokallio, T., Ojala, P., Nevalainen, T. J., and Nevalainen, O., (2000) Analysis of similarity of electrophoretic patterns in mRNA differential display. Electrophoresis 21(14), 2947–2956.

    Article  PubMed  CAS  Google Scholar 

  45. Korenberg, J., Chen, X., Adams, M., and Venter, J. (1995) Towards a cDNA map of the human genome. Genomics 29364–29370.

  46. Spinella, D., Bernadino, A., Redding, A., et al. (1999) Tandem arrayed ligation of expressed sequence tags (TALEST): a new method for generating global gene expression profiles. Nucleic Acids Research 27(18), e22.

    Google Scholar 

  47. Waller, H. A. and Savage, A. K. (1994) Analysis of gene transcription in situ: methodologica considerations and applications in pathology. J. Histotechnol. 17, 203–218.

    CAS  Google Scholar 

  48. Lewis, F. (1996). An approach to in situ PCR. PE Applied Biosystems, CA.

  49. Nuovo, G. J., Gallery, F., Hom, R., MacConnell, P., and Bloch, W. (1993) Importance of different variables for enhancing in situ detection of PCR-amplified DNA. PCR Methods Applic. 2, 305–312.

    CAS  Google Scholar 

  50. O’Leary, J. J., Browne, G., Landers, R. J., et al. (1994) The importance of fixation procedures on DNA template and its suitability for solution-phase polymerase chain reaction and PCR in situ hybridization. Histochem. J. 26, 337–346.

    Article  PubMed  CAS  Google Scholar 

  51. Waller, H. A. and Savage, A. K. (2000) mRNA detection by in situ RT-PCR. Chapter 46, pp. 417–429. In Ovarian Cancer: Methods and Protocols (Bartlett, J. M. S., ed.) Vol. 39 Methods in Molecular Medicine. Humana Press, Totowa, New Jersey, 0 USA.

    Chapter  Google Scholar 

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  1. University Department of Surgery, Level II Queen Elizabeth Building, Glasgow Royal Infirmary, G31 2ER, Glasgow, United Kingdom

    John M. S. Bartlett

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Bartlett, J.M.S. Approaches to the analysis of gene expression using mRNA. Mol Biotechnol 21, 149–160 (2002). https://doi.org/10.1385/MB:21:2:149

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