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Title: Use and Misuse of Cq in qPCR Data Analysis and Reporting
Authors: Ruiz-Villalba, Adrián
Ruijter, Jan M.
van den Hoff, Maurice J. B.
metadata.dc.contributor.authoraffiliation: [Ruiz-Villalba,A] Department of Animal Biology, Faculty of Sciences, Instituto Malagueño de Biomedicina (IBIMA), University of Málaga, Málaga, Spain. [Ruiz-Villalba,A] BIONAND, Centro Andaluz de Nanomedicina y Biotecnología, Junta de Andalucía, Universidad de Málaga, Málaga, Spain. [Ruijter,JM; van den Hoff, M.J.B] Department of Medical Biology, Amsterdam University Medical Centres, Location Academic Medical Center, Meibergdreef 15, Amsterdam, The Netherlands.
Keywords: qPCR analysis;Cq;Quantification cycle;Quantification threshold;PCR efficiency;Poisson variation;LOD;LOQ;Artefactos;Laboratorios;Expresión génica;Reacción en cadena en tiempo real de la polimerasa
metadata.dc.subject.mesh: Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Artifacts
Medical Subject Headings::Health Care::Health Care Facilities, Manpower, and Services::Health Facilities::Laboratories
Medical Subject Headings::Phenomena and Processes::Genetic Phenomena::Genetic Processes::Gene Expression
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Nucleic Acid Amplification Techniques::Polymerase Chain Reaction::Real-Time Polymerase Chain Reaction
Issue Date: 29-May-2021
Publisher: MDPI
Citation: Ruiz-Villalba A, Ruijter JM, van den Hoff MJB. Use and Misuse of Cq in qPCR Data Analysis and Reporting. Life. 2021 May 29;11(6):496
Abstract: In the analysis of quantitative PCR (qPCR) data, the quantification cycle (Cq) indicates the position of the amplification curve with respect to the cycle axis. Because Cq is directly related to the starting concentration of the target, and the difference in Cq values is related to the starting concentration ratio, the only results of qPCR analysis reported are often Cq, ΔCq or ΔΔCq values. However, reporting of Cq values ignores the fact that Cq values may differ between runs and machines, and, therefore, cannot be compared between laboratories. Moreover, Cq values are highly dependent on the PCR efficiency, which differs between assays and may differ between samples. Interpreting reported Cq values, assuming a 100% efficient PCR, may lead to assumed gene expression ratios that are 100-fold off. This review describes how differences in quantification threshold setting, PCR efficiency, starting material, PCR artefacts, pipetting errors and sampling variation are at the origin of differences and variability in Cq values and discusses the limits to the interpretation of observed Cq values. These issues can be avoided by calculating efficiency-corrected starting concentrations per reaction. The reporting of gene expression ratios and fold difference between treatments can then easily be based on these starting concentrations.
metadata.dc.identifier.doi: 10.3390/life11060496
ISSN: 2075-1729 (Online)
Appears in Collections:01- Artículos - BIONAND - Centro Andaluz de Nanomedicina y Biotecnología
01- Artículos - IBIMA. Instituto de Investigación Biomédica de Málaga

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