Nucleic Acids Research, 2001, Vol. 29, No. 9 e45
© 2001 Oxford University Press
A new mathematical model for relative quantification in real-time RT–PCR
Institute of Physiology, FML-Weihenstephan, Center of Life and Food Sciences, Technical University of Munich, Germany
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT–PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
* To whom correspondence should be addressed at present address: Institut für Physiologie, Weihenstephaner Berg 3, 85354 Freising, Weihenstephan, Germany. Tel: +49 8161 71 3511; Fax: +49 8161 71 4204; Email: pfaffl{at}weihenstephan.de
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A. Huggins, N. Paschalidis, R. J. Flower, M. Perretti, and F. D'Acquisto Annexin-1-deficient dendritic cells acquire a mature phenotype during differentiation FASEB J, April 1, 2009; 23(4): 985 - 996. [Abstract] [Full Text] [PDF]
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H. H. Pua, J. Guo, M. Komatsu, and Y.-W. He Autophagy Is Essential for Mitochondrial Clearance in Mature T Lymphocytes J. Immunol., April 1, 2009; 182(7): 4046 - 4055. [Abstract] [Full Text] [PDF]
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A. Meyer-Bahlburg, A. D. Bandaranayake, S. F. Andrews, and D. J. Rawlings Reduced c-myc Expression Levels Limit Follicular Mature B Cell Cycling in Response to TLR Signals J. Immunol., April 1, 2009; 182(7): 4065 - 4075. [Abstract] [Full Text] [PDF]
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A. J. Tipping, C. Pina, A. Castor, D. Hong, N. P. Rodrigues, L. Lazzari, G. E. May, S. E. W. Jacobsen, and T. Enver High GATA-2 expression inhibits human hematopoietic stem and progenitor cell function by effects on cell cycle Blood, March 19, 2009; 113(12): 2661 - 2672. [Abstract] [Full Text] [PDF]
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S. Kailasan Vanaja, T. M. Bergholz, and T. S. Whittam Characterization of the Escherichia coli O157:H7 Sakai GadE Regulon J. Bacteriol., March 15, 2009; 191(6): 1868 - 1877. [Abstract] [Full Text] [PDF]
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F.-M. Mbitikon-Kobo, M. Vocanson, M.-C. Michallet, M. Tomkowiak, A. Cottalorda, G. S. Angelov, C.-A. Coupet, S. Djebali, A. Marcais, B. Dubois, et al. Characterization of a CD44/CD122int Memory CD8 T Cell Subset Generated under Sterile Inflammatory Conditions J. Immunol., March 15, 2009; 182(6): 3846 - 3854. [Abstract] [Full Text] [PDF]
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Y. Hiyoshi, H. Kamohara, R. Karashima, N. Sato, Y. Imamura, Y. Nagai, N. Yoshida, E. Toyama, N. Hayashi, M. Watanabe, et al. MicroRNA-21 Regulates the Proliferation and Invasion in Esophageal Squamous Cell Carcinoma Clin. Cancer Res., March 15, 2009; 15(6): 1915 - 1922. [Abstract] [Full Text] [PDF]
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 A. D. Silk, A. J. Holland, and D. W. Cleveland Requirements for NuMA in maintenance and establishment of mammalian spindle poles J. Cell Biol., March 9, 2009; 184(5): 677 - 690. [Abstract] [Full Text] [PDF]
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G. Lucking, M. K. Dommel, S. Scherer, A. Fouet, and M. Ehling-Schulz Cereulide synthesis in emetic Bacillus cereus is controlled by the transition state regulator AbrB, but not by the virulence regulator PlcR Microbiology, March 1, 2009; 155(3): 922 - 931. [Abstract] [Full Text] [PDF]
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C. Herve, P. Dabos, C. Bardet, A. Jauneau, M. C. Auriac, A. Ramboer, F. Lacout, and D. Tremousaygue In Vivo Interference with AtTCP20 Function Induces Severe Plant Growth Alterations and Deregulates the Expression of Many Genes Important for Development Plant Physiology, March 1, 2009; 149(3): 1462 - 1477. [Abstract] [Full Text] [PDF]
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Y. Huo, S. C. McConnell, S.-R. Liu, R. Yang, T.-T. Zhang, C.-W. Sun, L.-C. Wu, and T. M. Ryan Humanized Mouse Model of Cooley's Anemia J. Biol. Chem., February 20, 2009; 284(8): 4889 - 4896. [Abstract] [Full Text] [PDF]
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J. B. Boonyaratanakornkit, E. J. Bartlett, E. Amaro-Carambot, P. L. Collins, B. R. Murphy, and A. C. Schmidt The C Proteins of Human Parainfluenza Virus Type 1 (HPIV1) Control the Transcription of a Broad Array of Cellular Genes That Would Otherwise Respond to HPIV1 Infection J. Virol., February 15, 2009; 83(4): 1892 - 1910. [Abstract] [Full Text] [PDF]
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C. B. Mathias, E.-J. Freyschmidt, B. Caplan, T. Jones, D. Poddighe, W. Xing, K. L. Harrison, M. F. Gurish, and H. C. Oettgen IgE Influences the Number and Function of Mature Mast Cells, but Not Progenitor Recruitment in Allergic Pulmonary Inflammation J. Immunol., February 15, 2009; 182(4): 2416 - 2424. [Abstract] [Full Text] [PDF]
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N. Jacob, H. Yang, L. Pricop, Y. Liu, X. Gao, S. G. Zheng, J. Wang, H.-X. Gao, C. Putterman, M. N. Koss, et al. Accelerated Pathological and Clinical Nephritis in Systemic Lupus Erythematosus-Prone New Zealand Mixed 2328 Mice Doubly Deficient in TNF Receptor 1 and TNF Receptor 2 via a Th17-Associated Pathway J. Immunol., February 15, 2009; 182(4): 2532 - 2541. [Abstract] [Full Text] [PDF]
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G. W. Prager, J. Mihaly, P. M. Brunner, Y. Koshelnick, G. Hoyer-Hansen, and B. R. Binder Urokinase mediates endothelial cell survival via induction of the X-linked inhibitor of apoptosis protein Blood, February 5, 2009; 113(6): 1383 - 1390. [Abstract] [Full Text] [PDF]
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C. Barreiro, D. Nakunst, A. T. Huser, H. D. de Paz, J. Kalinowski, and J. F. Martin Microarray studies reveal a 'differential response' to moderate or severe heat shock of the HrcA- and HspR-dependent systems in Corynebacterium glutamicum Microbiology, February 1, 2009; 155(2): 359 - 372. [Abstract] [Full Text] [PDF]
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N. Sturm, Y. Hu, H. Zimmermann, K. Fritz-Wolf, S. Wittlin, S. Rahlfs, and K. Becker Compounds Structurally Related to Ellagic Acid Show Improved Antiplasmodial Activity Antimicrob. Agents Chemother., February 1, 2009; 53(2): 622 - 630. [Abstract] [Full Text] [PDF]
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