Visualization of Gene Expression by Fluorescent Multiplex Reverse Transcriptase-PCR Amplification

  • María Rosa Ponce
  • Víctor Quesada
  • Andrea Hricová
  • José Luis Micol
Part of the Methods in Molecular Biology book series (MIMB, volume 353)


Many developmental and physiological analyses, population studies, and diagnostic tests can be performed by simply determining the presence or absence of a limited number of gene products. Here, we describe a rapid and sensitive procedure, based on the reverse transcription of total RNA samples followed by the co-amplification of specific complementary DNA molecules, for the simultaneous detection of different transcripts. Multiplex PCR amplification products are obtained in a single reaction mix containing several primer pairs, each of which includes a fluorescently labeled oligonucleotide; the amplification products are finally electrophoresed in an automated DNA sequencer controlled by fragment analysis software. The electropherograms obtained in this way allow a semiquantitative and efficient visualization of gene expression.

Key Words

Differential gene expression fluorescent labeling multiplex polymerase chain reaction transcription detection 


  1. 1.
    Alwine, J. C., Kemp, D. J., and Stark, G. R. (1997) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. USA 74, 5350–5354.CrossRefGoogle Scholar
  2. 2.
    Alwine, J. C., Kemp, D. J., Parker, B. A., et al. (1979) Detection of specific RNAs or specific fragments of DNA by fractionation in gels and transfer to diazobenzyloxymethyl paper. Methods Enzymol. 68, 220–242.PubMedCrossRefGoogle Scholar
  3. 3.
    Rappolee, D. A., Mark, D., Banda, M. J., and Werb, Z. (1988) Wound macrophages express TGF-alpha and other growth factors in vivo: analysis by mRNA phenotyping. Science 241, 708–712.PubMedCrossRefGoogle Scholar
  4. 4.
    Ramsay, G. (1998) DNA chips: state-of-the art. Nat. Biotechnol. 16, 40–44.PubMedCrossRefGoogle Scholar
  5. 5.
    Ponce, M. R., Pérez-Pérez, J. M., Piqueras, P., and Micol, J. L. (2000) A multiplex reverse transcriptase-polymerase chain reaction method for fluorescence-based semiautomated detection of gene expression in Arabidopsis thaliana. Planta 211, 606–608.PubMedCrossRefGoogle Scholar
  6. 6.
    Riechmann, J. L. and Meyerowitz, E. M. (1998) The AP2/EREBP family of plant transcription factors. Biol. Chem. 379, 633–646.PubMedCrossRefGoogle Scholar
  7. 7.
    Riechmann, J. L. and Meyerowitz, E. M. (1997) MADS domain proteins in plant development. Biol. Chem. 378, 1079–1101.PubMedCrossRefGoogle Scholar
  8. 8.
    Long, J. A., Moan, E. I., Medford, J. I., and Barton, M. K. (1996) A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis. Nature 379, 66–69.PubMedCrossRefGoogle Scholar
  9. 9.
    Kerstetter, R. A. and Poethig, R. S. (1998) The specification of leaf identity during shoot development. Annu. Rev. Cell Dev. Biol. 14, 373–398.PubMedCrossRefGoogle Scholar
  10. 10.
    Berná, G., Robles, P., and Micol, J. L. (1999) A mutational analysis of leaf morphogenesis in Arabidopsis thaliana. Genetics 152, 729–742.PubMedGoogle Scholar
  11. 11.
    Serrano-Cartagena J., Candela, H., Robles, P., et al. (2000) Genetic analysis of incurvata mutants reveals three independent genetic operations at work in Arabidopsis leaf morphogenesis. Genetics 156, 1363–1377.PubMedGoogle Scholar
  12. 12.
    Byrne, M. E., Barley, R., Curtis, M., et al. (2000) Asymmetric leaves1 mediates leaf patterning and stem cell function in Arabidopsis. Nature 408, 967–971.PubMedCrossRefGoogle Scholar
  13. 13.
    Quesada, V., Ponce, M. R., and Micol, J. L. (1999) OTC and AUL1, two convergent and overlapping genes in the nuclear genome of Arabidopsis thaliana. FEBS Lett. 461, 101–106.PubMedCrossRefGoogle Scholar
  14. 14.
    Wallace, R. B., Shaffer, J., Murphy, R. F., Bonner, J., Hirose, T., and Itakura, K. (1979) Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch. Nucleic Acids Res. 6, 3543–3557.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • María Rosa Ponce
    • 1
    • 2
  • Víctor Quesada
    • 1
    • 2
  • Andrea Hricová
    • 1
    • 2
  • José Luis Micol
    • 1
    • 2
  1. 1.División de GenéticaUniversidad Miguel Hernández, Campus de ElcheAlicanteSpain
  2. 2.Instituto de BioingenieríaUniversidad Miguel Hernández, Campus de ElcheAlicanteSpain

Personalised recommendations