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Quantitative RT-PCR: Pitfalls and Potential
Open accessAbstract
Reverse transcription PCR (RT-PCR) represents a sensitive and powerful tool for analyzing RNA. While it has tremendous potential for quantitative applications, a comprehensive knowledge of its technical aspects is required. Successful quantitative RT-PCR involves correction for experimental variations in individual RT and PCR efficiencies. This review addresses the mathematics of RT-PCR, choice of RNA standards (internal vs. external) and quantification strategies (competitive, noncompetitive and kinetic [real-time] amplification). Finally, the discussion turns to practical considerations in experimental design. It is hoped that this review will be appropriate for those undertaking these experiments for the first time or wishing to improve (or validate) a technique in what is frequently a confusing and contradictory field.
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Citations
Cited by
A new mathematical model for relative quantification in real-time RT-PCR
Pfaffl, M. W. (2001) · Nucleic Acids Research · doi:10.1093/nar/29.9.e45 · Cited by 34,610
Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data
Ruijter, J. M., Ramakers, C., Hoogaars, W. M. H., Karlen, Y., Bakker, O., van den Hoff, M. J. B., & Moorman, A. F. M. (2009) · Nucleic Acids Research · doi:10.1093/nar/gkp045 · Cited by 3,043
Twenty-Five Years of Quantitative PCR for Gene Expression Analysis
VanGuilder, H. D., Vrana, K. E., & Freeman, W. M. (2008) · BioTechniques · doi:10.2144/000112776 · Cited by 1,246
Self-citationde Kok, J. B., Roelofs, R. W., Giesendorf, B. A., Pennings, J. L., Waas, E. T., Feuth, T., Swinkels, D. W., & Span, P. N. (2005) · Laboratory Investigation · doi:10.1038/labinvest.3700208 · Cited by 580
Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays
Yuen, T. (2002) · Nucleic Acids Research · doi:10.1093/nar/30.10.e48 · Cited by 520
Metadata
- Authors
- Freeman, W. M., Walker, S. J., & Vrana, K. E.
- Venue
- BioTechniques
- Publisher
- Informa UK Limited
- Published
- 1999-01
- Volume
- 26
- Issue
- 1
- Pages
- 112-125
- ISSN
- 0736-6205 (ISSN-L)
- 1940-9818
- OpenAlex
- W1569955072
- Cited by
- 1,147
- References
- 0
- FWCI
- 21.86
- Type
- journal article
- Language
- en
- Open access
- gold
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