Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data
Open accessAbstract
Despite the central role of quantitative PCR (qPCR) in the quantification of mRNA transcripts, most analyses of qPCR data are still delegated to the software that comes with the qPCR apparatus. This is especially true for the handling of the fluorescence baseline. This article shows that baseline estimation errors are directly reflected in the observed PCR efficiency values and are thus propagated exponentially in the estimated starting concentrations as well as 'fold-difference' results. Because of the unknown origin and kinetics of the baseline fluorescence, the fluorescence values monitored in the initial cycles of the PCR reaction cannot be used to estimate a useful baseline value. An algorithm that estimates the baseline by reconstructing the log-linear phase downward from the early plateau phase of the PCR reaction was developed and shown to lead to very reproducible PCR efficiency values. PCR efficiency values were determined per sample by fitting a regression line to a subset of data points in the log-linear phase. The variability, as well as the bias, in qPCR results was significantly reduced when the mean of these PCR efficiencies per amplicon was used in the calculation of an estimate of the starting concentration per sample.
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The mean efficiency of the amplicons was calculated by Linreg v. 12.1 based on the log linear phase of the amplification curve.
“The mean efficiency of the amplicons was calculated by the program Linreg v. 12.1 based on the log linear phase of the amplification curve [51].”
From Relative expression of genes of terpene metabolism in different tissues of Artemisia annuaL (2011) · cites this paper as [51]
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- Authors
- Ruijter, J. M., Ramakers, C., Hoogaars, W. M. H., Karlen, Y., Bakker, O., van den Hoff, M. J. B., & Moorman, A. F. M.
- Affiliations
- Amsterdam UMC Location University of Amsterdam (NL)
- Maastricht University (NL)
- Corresponding author
- Ruijter, J. M.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2009-02-22
- Volume
- 37
- Issue
- 6
- Pages
- e45-e45
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC2665230
- OpenAlex
- W2136309995
- Cited by
- 3,043
- References
- 32
- Citations by year
- 2026: 113 · 2025: 136 · 2024: 140 · 2023: 156 · 2022: 194 · 2021: 291 · 2020: 271 · 2019: 235 · 2018: 225 · 2017: 196 · 2016: 216 · 2015: 205 · 2014: 164 · 2013: 158 · 2012: 139
- FWCI
- 45.85
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Nucleic Acids Research (publishedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- SERVAL (Université de Lausanne) (publishedVersion, other-oa)
- UvA-DARE (University of Amsterdam) (submittedVersion, other-oa)
- IRIS (submittedVersion, cc-by-nc)
- Data Archiving and Networked Services (DANS) (submittedVersion)
- UvA-DARE (University of Amsterdam) (submittedVersion, cc-by-nc)
- Pure Amsterdam UMC (submittedVersion)
- Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
- Funders
- European Commission
- Universiteit Leiden
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene expression and cancer classification
- Health Sciences › Medicine › Physiology › Clinical Laboratory Practices and Quality Control
- Keywords
- real-time PCR, PCR efficiency, baseline estimation, mRNA quantification
- MeSH terms
- Algorithms, Animals, Chick Embryo, Fluorescence, Linear Models, Reverse Transcriptase Polymerase Chain Reaction
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