Abstract
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.
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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.
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.
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.
Ramsay, G. (1998) DNA chips: state-of-the art. Nat. Biotechnol. 16, 40–44.
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.
Riechmann, J. L. and Meyerowitz, E. M. (1998) The AP2/EREBP family of plant transcription factors. Biol. Chem. 379, 633–646.
Riechmann, J. L. and Meyerowitz, E. M. (1997) MADS domain proteins in plant development. Biol. Chem. 378, 1079–1101.
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.
Kerstetter, R. A. and Poethig, R. S. (1998) The specification of leaf identity during shoot development. Annu. Rev. Cell Dev. Biol. 14, 373–398.
Berná, G., Robles, P., and Micol, J. L. (1999) A mutational analysis of leaf morphogenesis in Arabidopsis thaliana. Genetics 152, 729–742.
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.
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.
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.
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.
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© 2007 Humana Press Inc.
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Ponce, M.R., Quesada, V., Hricová, A., Micol, J.L. (2007). Visualization of Gene Expression by Fluorescent Multiplex Reverse Transcriptase-PCR Amplification. In: Hilario, E., Mackay, J. (eds) Protocols for Nucleic Acid Analysis by Nonradioactive Probes. Methods in Molecular Biology, vol 353. Humana Press. https://doi.org/10.1385/1-59745-229-7:143
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DOI: https://doi.org/10.1385/1-59745-229-7:143
Publisher Name: Humana Press
Print ISBN: 978-1-58829-430-2
Online ISBN: 978-1-59745-229-8
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