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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

Michael W. Pfaffl*

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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Genome Res., August 1, 2009; 19(8): 1462 - 1470.
[Abstract] [Full Text] [PDF]


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Eukaryot CellHome page
B. Birkaya, A. Maddi, J. Joshi, S. J. Free, and P. J. Cullen
Role of the Cell Wall Integrity and Filamentous Growth Mitogen-Activated Protein Kinase Pathways in Cell Wall Remodeling during Filamentous Growth
Eukaryot. Cell, August 1, 2009; 8(8): 1118 - 1133.
[Abstract] [Full Text] [PDF]


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Am. J. Physiol. Endocrinol. Metab.Home page
R. Comitato, K. Nesaretnam, G. Leoni, R. Ambra, R. Canali, A. Bolli, M. Marino, and F. Virgili
A novel mechanism of natural vitamin E tocotrienol activity: involvement of ER{beta} signal transduction
Am J Physiol Endocrinol Metab, August 1, 2009; 297(2): E427 - E437.
[Abstract] [Full Text] [PDF]


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Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
S. L. Steele, K. H. A. Lo, V. W. T. Li, S. H. Cheng, M. Ekker, and S. F. Perry
Loss of M2 muscarinic receptor function inhibits development of hypoxic bradycardia and alters cardiac {beta}-adrenergic sensitivity in larval zebrafish (Danio rerio)
Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2009; 297(2): R412 - R420.
[Abstract] [Full Text] [PDF]


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Cancer Prevention ResearchHome page
A. Giros, M. Grzybowski, V. R. Sohn, E. Pons, J. Fernandez-Morales, R. M. Xicola, P. Sethi, J. Grzybowski, A. Goel, C. R. Boland, et al.
Regulation of Colorectal Cancer Cell Apoptosis by the n-3 Polyunsaturated Fatty Acids Docosahexaenoic and Eicosapentaenoic
Cancer Prevention Research, August 1, 2009; 2(8): 732 - 742.
[Abstract] [Full Text] [PDF]


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GeneticsHome page
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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Plant CellHome page
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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Plant CellHome page
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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BloodHome page
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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Proc. Natl. Acad. Sci. USAHome page
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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J. Neurosci.Home page
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. Immunol.Home page
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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Mol. Biol. CellHome page
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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Drug Metab. Dispos.Home page
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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Nucleic Acids ResHome page
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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J. Bacteriol.Home page
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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J. Bacteriol.Home page
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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Appl. Environ. Microbiol.Home page
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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Mol PlantHome page
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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Cereb CortexHome page
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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FASEB J.Home page
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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FASEB J.Home page
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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Eukaryot CellHome page
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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Am. J. Pathol.Home page
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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Molecular Cancer TherapeuticsHome page
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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Plant Physiol.Home page
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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J. Immunol.Home page
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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J. Immunol.Home page
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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