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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 D. E. Davis, H. C. Roh, K. Deshmukh, J. J. Bruinsma, D. L. Schneider, J. Guthrie, J. D. Robertson, and K. Kornfeld The Cation Diffusion Facilitator Gene cdf-2 Mediates Zinc Metabolism in Caenorhabditis elegans Genetics, August 1, 2009; 182(4): 1015 - 1033. [Abstract] [Full Text] [PDF]
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 J. Zhao and R. A. Dixon MATE Transporters Facilitate Vacuolar Uptake of Epicatechin 3'-O-Glucoside for Proanthocyanidin Biosynthesis in Medicago truncatula and Arabidopsis PLANT CELL, August 1, 2009; 21(8): 2323 - 2340. [Abstract] [Full Text] [PDF]
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P. Pesaresi, A. Hertle, M. Pribil, T. Kleine, R. Wagner, H. Strissel, A. Ihnatowicz, V. Bonardi, M. Scharfenberg, A. Schneider, et al. Arabidopsis STN7 Kinase Provides a Link between Short- and Long-Term Photosynthetic Acclimation PLANT CELL, August 1, 2009; 21(8): 2402 - 2423. [Abstract] [Full Text] [PDF]
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M. Remke, S. Pfister, C. Kox, G. Toedt, N. Becker, A. Benner, W. Werft, S. Breit, S. Liu, F. Engel, et al. High-resolution genomic profiling of childhood T-ALL reveals frequent copy-number alterations affecting the TGF-{beta} and PI3K-AKT pathways and deletions at 6q15-16.1 as a genomic marker for unfavorable early treatment response Blood, July 30, 2009; 114(5): 1053 - 1062. [Abstract] [Full Text] [PDF]
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V. T. Samuel, S. A. Beddow, T. Iwasaki, X.-M. Zhang, X. Chu, C. D. Still, G. S. Gerhard, and G. I. Shulman Fasting hyperglycemia is not associated with increased expression of PEPCK or G6Pc in patients with Type 2 Diabetes PNAS, July 21, 2009; 106(29): 12121 - 12126. [Abstract] [Full Text] [PDF]
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L. P. Cacheaux, S. Ivens, Y. David, A. J. Lakhter, G. Bar-Klein, M. Shapira, U. Heinemann, A. Friedman, and D. Kaufer Transcriptome Profiling Reveals TGF-{beta} Signaling Involvement in Epileptogenesis J. Neurosci., July 15, 2009; 29(28): 8927 - 8935. [Abstract] [Full Text] [PDF]
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J. W. Leavenworth, X. Ma, Y.-y. Mo, and M. E. Pauza SUMO Conjugation Contributes to Immune Deviation in Nonobese Diabetic Mice by Suppressing c-Maf Transactivation of IL-4 J. Immunol., July 15, 2009; 183(2): 1110 - 1119. [Abstract] [Full Text] [PDF]
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N. L. Quach, S. Biressi, L. F. Reichardt, C. Keller, and T. A. Rando Focal Adhesion Kinase Signaling Regulates the Expression of Caveolin 3 and {beta}1 Integrin, Genes Essential for Normal Myoblast Fusion Mol. Biol. Cell, July 15, 2009; 20(14): 3422 - 3435. [Abstract] [Full Text] [PDF]
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S. Helmig, B. Hadzaad, J. Dohrel, and J. Schneider Influence of the Cyp1B1 L432V Gene Polymorphism and Exposure to Tobacco Smoke on Cyp1B1 mRNA Expression in Human Leukocytes Drug Metab. Dispos., July 1, 2009; 37(7): 1490 - 1495. [Abstract] [Full Text] [PDF]
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L. Lin, S. Liu, H. Brockway, J. Seok, P. Jiang, W. H. Wong, and Y. Xing Using high-density exon arrays to profile gene expression in closely related species Nucleic Acids Res., July 1, 2009; 37(12): e90 - e90. [Abstract] [Full Text] [PDF]
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S. Donat, K. Streker, T. Schirmeister, S. Rakette, T. Stehle, M. Liebeke, M. Lalk, and K. Ohlsen Transcriptome and Functional Analysis of the Eukaryotic-Type Serine/Threonine Kinase PknB in Staphylococcus aureus J. Bacteriol., July 1, 2009; 191(13): 4056 - 4069. [Abstract] [Full Text] [PDF]
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S. Metz, A. Jager, and G. Klug In Vivo Sensitivity of Blue-Light-Dependent Signaling Mediated by AppA/PpsR or PrrB/PrrA in Rhodobacter sphaeroides J. Bacteriol., July 1, 2009; 191(13): 4473 - 4477. [Abstract] [Full Text] [PDF]
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S. Nouaille, S. Even, C. Charlier, Y. Le Loir, M. Cocaign-Bousquet, and P. Loubiere Transcriptomic Response of Lactococcus lactis in Mixed Culture with Staphylococcus aureus Appl. Envir. Microbiol., July 1, 2009; 75(13): 4473 - 4482. [Abstract] [Full Text] [PDF]
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J.-R. Wang, H. Hu, G.-H. Wang, J. Li, J.-Y. Chen, and P. Wu Expression of PIN Genes in Rice (Oryza sativa L.): Tissue Specificity and Regulation by Hormones Mol Plant, July 1, 2009; 2(4): 823 - 831. [Abstract] [Full Text] [PDF]
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 G. Curia, T. Papouin, P. Seguela, and M. Avoli Downregulation of Tonic GABAergic Inhibition in a Mouse Model of Fragile X Syndrome Cereb Cortex, July 1, 2009; 19(7): 1515 - 1520. [Abstract] [Full Text] [PDF]
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K.-D. Yu, G.-H. Di, L. Fan, J. Wu, Z. Hu, Z.-Z. Shen, W. Huang, and Z.-M. Shao A functional polymorphism in the promoter region of GSTM1 implies a complex role for GSTM1 in breast cancer FASEB J, July 1, 2009; 23(7): 2274 - 2287. [Abstract] [Full Text] [PDF]
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D. Urich, S. Soberanes, Z. Burgess, S. E. Chiarella, A. J. Ghio, K. M. Ridge, D. W. Kamp, N. S. Chandel, G. M. Mutlu, and G. R. S. Budinger Proapoptotic Noxa is required for particulate matter-induced cell death and lung inflammation FASEB J, July 1, 2009; 23(7): 2055 - 2064. [Abstract] [Full Text] [PDF]
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C. A. Durkin, T. Mock, and E. V. Armbrust Chitin in Diatoms and Its Association with the Cell Wall Eukaryot. Cell, July 1, 2009; 8(7): 1038 - 1050. [Abstract] [Full Text] [PDF]
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 S. Kempe, P. Heinz, E. Kokai, O. Devergne, N. Marx, and T. Wirth Epstein-Barr Virus-Induced Gene-3 Is Expressed in Human Atheroma Plaques Am. J. Pathol., July 1, 2009; 175(1): 440 - 447. [Abstract] [Full Text] [PDF]
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 J. Loeffler-Ragg, D. Mueller, G. Gamerith, T. Auer, S. Skvortsov, B. Sarg, I. Skvortsova, K. J. Schmitz, H.-J. Martin, J. Krugmann, et al. Proteomic identification of aldo-keto reductase AKR1B10 induction after treatment of colorectal cancer cells with the proteasome inhibitor bortezomib Mol. Cancer Ther., July 1, 2009; 8(7): 1995 - 2006. [Abstract] [Full Text] [PDF]
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F. Rahmani, M. Hummel, J. Schuurmans, A. Wiese-Klinkenberg, S. Smeekens, and J. Hanson Sucrose Control of Translation Mediated by an Upstream Open Reading Frame-Encoded Peptide Plant Physiology, July 1, 2009; 150(3): 1356 - 1367. [Abstract] [Full Text] [PDF]
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T. Rose, O. Lambotte, C. Pallier, J.-F. Delfraissy, and J.-H. Colle Identification and Biochemical Characterization of Human Plasma Soluble IL-7R: Lower Concentrations in HIV-1-Infected Patients J. Immunol., June 15, 2009; 182(12): 7389 - 7397. [Abstract] [Full Text] [PDF]
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C. van der Putten, E. A. Zuiderwijk-Sick, L. van Straalen, E. D. de Geus, L. A. Boven, I. Kondova, A. P. IJzerman, and J. J. Bajramovic Differential Expression of Adenosine A3 Receptors Controls Adenosine A2A Receptor-Mediated Inhibition of TLR Responses in Microglia J. Immunol., June 15, 2009; 182(12): 7603 - 7612. [Abstract] [Full Text] [PDF]
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