Abstract
Reverse transcriptase-polymerase chain reaction (RT-PCR) is a highly sensitive method that permits the detection and quantification of RNA transcripts. It is particularly useful where transcripts are in very low abundance and where the amount of starting RNA is fairly limited, such as from a protoplast transfection. The procedure is based on production of a cDNA copy of the transcript by primer extension (see Chapter 21) and PCR amplification of the cDNA transcript by Taq DNA polymerase. The ability to amplify cDNA transcripts by Taq polymerase gives the flexibility and sensitivity, found with standard DNA PCR amplification, in the analysis of RNAs.
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Simpson, C. G., Sinibaldi, R., and Brown, J. W. S. (1992) Rapid analysis of plant gene expression by a novel reverse transcriptase-PCR method. Plant J. 2, 835–836.
Golde, T. E., Estus, S., Usiak, M., Younkin, L. M., and Younkin, S. G. (1991) Expression of β-amyloid protein precursor mRNAs recognition of a novel alternatively spliced form and quantitation in Alzheimer’s disease using PCR. Neuron 4, 253–267.
Brown, J. W. S., Simpson, C. G., Simpson, G. G., Turnbull-Ross, A. D., and Clark, G. P. (1993) Plant pre-RNA splicing and splicing components. Phil. Trans. R. Soc Lond. B 342, 217–224.
Simpson C. G., Sawbridge, T. I., Jenkins, G. I., and Brown, J. W. S. (1992) Expression analysis of multigene families by RT-PCR. Nucleic Acids Res. 20, 5861–5862.
Brown, J. W. S., Simpson, G. G., Clark, G., Lyon, J., Kumar, A., and Simpson, C. G. (1993) Detection of antisense transcripts in transgenic plants by RT-PCR. Plant J. 4, 833–885.
Vaux, P., Guerineau, F., Waugh, R., and Brown, J. W. S. (1992) Characterization and expression of U1snRNA genes from potato. Plant Mol. Biol. 19, 959–971.
Hedley, P. E., Machray, G. C., Davies, H. V., Burch, L., and Waugh, R. (1994) Potato (Solanum tuberosum) invertase-encoding cDNAs and their differential expression. Gene 145, 211–214.
Chomczynski, P. and Sacchi, N. (1987) Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.
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© 1995 Humana Press Inc., Totowa, NJ
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Simpson, C.G., Brown, J.W.S. (1995). Applications of RT-PCR. In: Jones, H. (eds) Plant Gene Transfer and Expression Protocols. Methods in Molecular Biology™, vol 49. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-321-X:257
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DOI: https://doi.org/10.1385/0-89603-321-X:257
Publisher Name: Springer, Totowa, NJ
Print ISBN: 978-0-89603-321-4
Online ISBN: 978-1-59259-536-5
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