Abstract
Quantitative PCR (qPCR) has entered widespread use with the increasing availability of real-time PCR. By the incorporation of fluorescent dyes in the reaction mixture, increases in amplification products can be monitored throughout the reaction, enabling measurements to be taken in the exponential phase of the reaction, before the reaction plateau. Whatever the platform or chemistry involved, the starting point of a real-time assay is a tissue-specific RNA and the end point of a real-time reaction is an amplification plot. As such, rather than focusing on specific platforms or chemistries, herein we address the basic principles that underlie sample preparation, experimental design, use of internal controls, assay considerations, and approaches to data analysis.
The advent of real-time PCR has enabled high-throughput analysis of multiple transcripts from small tissue samples, with an unparalleled dynamic range and sensitivity. However, to new users, this technique may seem to require extensive optimization and troubleshooting to obtain reliable data; this is further compounded by the mass of technical variations present throughout the literature. The aim of this article is to provide the necessary basics to get a quantitative real-time PCR assay up and running, and to address some of the problems that may arise and how these may be resolved.
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References
Mullis, K., and Faloona, F. A. (1987) Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction. Meth. Enzymol. 255, 335–350.
Kainz, P. (2000) The PCR plateau phase—towards an understanding of its limitations. Biochim. Biophys. Acta 1494, 23–27.
Freeman, W. M., Walker, S. J., and Vrana, K. E. (1999) Quantitative RT-PCR: pitfalls and potential. Biotechniques 26, 112–122, 124–125.
Bustin, S.A. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 169–193.
Ginzinger, D. G. (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp. Hematol. 30, 503–512.
Klein, D. (2002) Quantification using real-time PCR technology: applications and limitations. Trends Mol. Med. 8, 257–260.
Walker, N. J. (2002) Tech.Sight. A technique whose time has come. Science. 296, 557–559.
Nadon, R., and Shoemaker, J. (2000) Statistical issues with microarrays: processing and analysis. Trends Genet. 18, 265–271.
Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (NY) 11, 1026–1030.
Higuchi, R., and Watson, R. (1999) Kinetic PCR analysis using a CCD camera and without using oligonucleotide probes. In: PCR Applications: Protocols for Functional Genomics (Innis, M. A., Gelfand, D. H., and Sninsky, J. J., eds.). Academic Press, San Diego, CA, pp. 263–284.
Bustin, S. A. (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 29, 23–39.
Stahlberg, A., Hakansson, J., Xian, X., Semb, H., and Kubista, M. (2004) Properties of the reverse transcription reaction in mRNA quantification. Clin. Chem. 50, 509–515.
Peirson, S. N., Butler, J. B., and Foster, R. G. (2003) Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids Res. 15, e73.
Ririe, K. M., Rasmussen, R. P., and Wittwer, C. T. (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 245, 154–160.
Vandesompele, J., De Preter, K., Pattyn, F., et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, research0034.1-0034.11
Livak, K. J. (1997) ABI Prism 7700 Sequence Detection System, in User Bulletin #2. PE Applied Biosystems.
Livak, K. J., and Schmittgen, T. W. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods. 25, 402–408.
Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, 2002–2007.
Liu, W., and Saint, D. A. (2002). A new quantitative method of real-time RT-PCR assay based on simulation of PCR kinetics. Anal. Biochem. 302, 52–59.
Liu, W., and Saint, D. (2002) Validation of a quantitative method for real time PCR kinetics. Biochem. Biophys. Res. Comm. 294, 347–353.
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Peirson, S.N., Butler, J.N. (2007). Quantitative Polymerase Chain Reaction. In: Rosato, E. (eds) Circadian Rhythms. Methods in Molecular Biology™, vol 362. Humana Press. https://doi.org/10.1007/978-1-59745-257-1_25
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DOI: https://doi.org/10.1007/978-1-59745-257-1_25
Publisher Name: Humana Press
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