How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments
Open accessAbstract
We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1-16 qPCR replicates per concentration and we tested 2-10 μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3-4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.
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Relative primer binding efficiencies were assessed through serial dilutions of cDNA.
“We first assessed relative primer binding efficiencies through a series of 4 (1:10) serial dilutions of an aliquot of cDNA as in [47].”
From Denisovan introgression has shaped the immune system of present-day Papuans (2022) · cites this paper as [47]
The Introduction describes estimating PCR amplification efficiency from standard curves made by serial dilution, and Methods > Preparation of standard curves reports sequential 10-fold dilutions of cDNA. The paper does not describe this as a measure of primer-binding efficiency. Its own series used five dilution steps, rather than the citing sentence’s four. (LLM, conf=0.85)
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Denisovan introgression has shaped the immune system of present-day Papuans
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- Authors
- Svec, D., Tichopad, A., Novosadova, V., Pfaffl, M. W., & Kubista, M.
- Affiliations
- Technical University of Munich (DE)
- Czech Academy of Sciences (CZ)
- Venue
- Biomolecular Detection and Quantification
- Publisher
- Elsevier BV
- Published
- 2015-03
- Volume
- 3
- Pages
- 9-16
- ISSN
- 2214-7535 (ISSN-L)
- PMC
- PMC4822216
- OpenAlex
- W2149903385
- Cited by
- 601
- References
- 34
- Citations by year
- 2026: 56 · 2025: 60 · 2024: 78 · 2023: 83 · 2022: 67 · 2021: 66 · 2020: 56 · 2019: 39 · 2018: 46 · 2017: 24 · 2016: 19 · 2015: 6
- FWCI
- 13.67
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- Biomolecular Detection and Quantification (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- ASEP (submittedVersion, public-domain)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Funders
- Grantová Agentura České Republiky (CZ.1.05/1.1.00/02.0109, AV0Z50520701, BIOCEV CZ.1.05/1.1.00/02.0109)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Social Sciences › Decision Sciences › Statistics, Probability and Uncertainty › Scientific Measurement and Uncertainty Evaluation
- Health Sciences › Medicine › Physiology › Clinical Laboratory Practices and Quality Control
- Keywords
- qPCR standard curve, technical replicates, sampling error, Monte Carlo simulation, dynamic range
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