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Nucleic Acids Research, 2002, Vol. 30, No. 9 e36
© 2002 Oxford University Press

Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR

Michael W. Pfaffl*, Graham W. Horgan1 and Leo Dempfle2

Institute of Physiology, FML-Weihenstephan, Center of Life and Food Sciences, Technical University of Munich, Germany, 1Biomathematics and Statistics Scotland, Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK and 2Department of Animal Science, Center of Life and Food Sciences, Technical University of Munich, Germany

Real-time reverse transcription followed by polymerase chain reaction (RT–PCR) is the most suitable method for the detection and quantification of mRNA. It offers high sensitivity, good reproducibility and a wide quantification range. Today, relative expression is increasingly used, where the expression of a target gene is standardised by a non-regulated reference gene. Several mathematical algorithms have been developed to compute an expression ratio, based on real-time PCR efficiency and the crossing point deviation of an unknown sample versus a control. But all published equations and available models for the calculation of relative expression ratio allow only for the determination of a single transcription difference between one control and one sample. Therefore a new software tool was established, named REST© (relative expression software tool), which compares two groups, with up to 16 data points in a sample and 16 in a control group, for reference and up to four target genes. The mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the sample and control group. Subsequently, the expression ratio results of the four investigated transcripts are tested for significance by a randomisation test. Herein, development and application of REST© is explained and the usefulness of relative expression in real-time PCR using REST© is discussed. The latest software version of REST© and examples for the correct use can be downloaded at http://www.wzw.tum.de/gene-quantification/.

* To whom correspondence should be addressed at present address: Institute of Physiology, FML-Weihenstephan, Life Science Zentrum Weihenstephan, Technische Universitaet Muenchen, Weihenstephaner Berg 3, D-85350 Freising–Weihenstephan, Germany. Tel: +49 8161 71 3511; Fax: +49 8161 71 4204; Email: pfaffl{at}wzw.tum.de


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Adenosine 5'-Monophosphate-Activated Protein Kinase Promotes Macrophage Polarization to an Anti-Inflammatory Functional Phenotype
J. Immunol., December 15, 2008; 181(12): 8633 - 8641.
[Abstract] [Full Text] [PDF]


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Proc. Natl. Acad. Sci. USAHome page
K. R. Reed, D. Athineos, V. S. Meniel, J. A. Wilkins, R. A. Ridgway, Z. D. Burke, V. Muncan, A. R. Clarke, and O. J. Sansom
B-catenin deficiency, but not Myc deletion, suppresses the immediate phenotypes of APC loss in the liver
PNAS, December 2, 2008; 105(48): 18919 - 18923.
[Abstract] [Full Text] [PDF]


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Cardiovasc ResHome page
P. Sasse, D. Malan, M. Fleischmann, W. Roell, E. Gustafsson, T. Bostani, Y. Fan, T. Kolbe, M. Breitbach, K. Addicks, et al.
Perlecan is critical for heart stability
Cardiovasc Res, December 1, 2008; 80(3): 435 - 444.
[Abstract] [Full Text] [PDF]


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J. Bacteriol.Home page
F. Hommais, C. Oger-Desfeux, F. Van Gijsegem, S. Castang, S. Ligori, D. Expert, W. Nasser, and S. Reverchon
PecS Is a Global Regulator of the Symptomatic Phase in the Phytopathogenic Bacterium Erwinia chrysanthemi 3937
J. Bacteriol., November 15, 2008; 190(22): 7508 - 7522.
[Abstract] [Full Text] [PDF]


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J Exp BotHome page
A. Zamboni, L. Minoia, A. Ferrarini, G. B. Tornielli, E. Zago, M. Delledonne, and M. Pezzotti
Molecular analysis of post-harvest withering in grape by AFLP transcriptional profiling
J. Exp. Bot., November 13, 2008; (2008) ern256v1.
[Abstract] [Full Text] [PDF]


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Hum Mol GenetHome page
C.-H. Chan, H. M. Mitchison, and D. A. Pearce
Transcript and in silico analysis of CLN3 in juvenile neuronal ceroid lipofuscinosis and associated mouse models
Hum. Mol. Genet., November 1, 2008; 17(21): 3332 - 3339.
[Abstract] [Full Text] [PDF]


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Exp. Biol. Med.Home page
G. N. Girgis, S. Sharif, J. R. Barta, H. J. Boermans, and T. K. Smith
Immunomodulatory Effects of Feed-Borne Fusarium Mycotoxins in Chickens Infected with Coccidia
Experimental Biology and Medicine, November 1, 2008; 233(11): 1411 - 1420.
[Abstract] [Full Text] [PDF]


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J. Am. Soc. Nephrol.Home page
J. Ho, K. H. Ng, S. Rosen, A. Dostal, R. I. Gregory, and J. A. Kreidberg
Podocyte-Specific Loss of Functional MicroRNAs Leads to Rapid Glomerular and Tubular Injury
J. Am. Soc. Nephrol., November 1, 2008; 19(11): 2069 - 2075.
[Abstract] [Full Text] [PDF]


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J. Gen. Virol.Home page
V. Dell'Oste, B. Azzimonti, M. Mondini, M. De Andrea, C. Borgogna, R. Mesturini, R. Accardi, M. Tommasino, S. Landolfo, U. Dianzani, et al.
Altered expression of UVB-induced cytokines in human papillomavirus-immortalized epithelial cells
J. Gen. Virol., October 1, 2008; 89(10): 2461 - 2466.
[Abstract] [Full Text] [PDF]


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J. Gen. Virol.Home page
P. J. Dufresne, E. Ubalijoro, M. G. Fortin, and J.-F. Laliberte
Arabidopsis thaliana class II poly(A)-binding proteins are required for efficient multiplication of turnip mosaic virus
J. Gen. Virol., September 1, 2008; 89(9): 2339 - 2348.
[Abstract] [Full Text] [PDF]


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Infect. Immun.Home page
M. E. Embers, X. Alvarez, T. Ooms, and M. T. Philipp
The Failure of Immune Response Evasion by Linear Plasmid 28-1-Deficient Borrelia burgdorferi Is Attributable to Persistent Expression of an Outer Surface Protein
Infect. Immun., September 1, 2008; 76(9): 3984 - 3991.
[Abstract] [Full Text] [PDF]


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GeneticsHome page
J. Kamerewerd, M. Jansson, M. Nowrousian, S. Poggeler, and U. Kuck
Three {alpha}-Subunits of Heterotrimeric G Proteins and an Adenylyl Cyclase Have Distinct Roles in Fruiting Body Development in the Homothallic Fungus Sordaria macrospora
Genetics, September 1, 2008; 180(1): 191 - 206.
[Abstract] [Full Text] [PDF]


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Cardiovasc ResHome page
R. Bauer, G.A. MacGowan, A. Blain, K. Bushby, and V. Straub
Steroid treatment causes deterioration of myocardial function in the {delta}-sarcoglycan-deficient mouse model for dilated cardiomyopathy
Cardiovasc Res, September 1, 2008; 79(4): 652 - 661.
[Abstract] [Full Text] [PDF]


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J. Biol. Chem.Home page
I. Tchivilev, N. R. Madamanchi, A. E. Vendrov, X.-L. Niu, and M. S. Runge
Identification of a Protective Role for Protein Phosphatase 1c{gamma}1 against Oxidative Stress-induced Vascular Smooth Muscle Cell Apoptosis
J. Biol. Chem., August 8, 2008; 283(32): 22193 - 22205.
[Abstract] [Full Text] [PDF]


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GeneticsHome page
R. R. Bellone, S. A. Brooks, L. Sandmeyer, B. A. Murphy, G. Forsyth, S. Archer, E. Bailey, and B. Grahn
Differential Gene Expression of TRPM1, the Potential Cause of Congenital Stationary Night Blindness and Coat Spotting Patterns (LP) in the Appaloosa Horse (Equus caballus)
Genetics, August 1, 2008; 179(4): 1861 - 1870.
[Abstract] [Full Text] [PDF]


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Drug Metab. Dispos.Home page
C. MacLean, U. Moenning, A. Reichel, and G. Fricker
Closing the Gaps: A Full Scan of the Intestinal Expression of P-Glycoprotein, Breast Cancer Resistance Protein, and Multidrug Resistance-Associated Protein 2 in Male and Female Rats
Drug Metab. Dispos., July 1, 2008; 36(7): 1249 - 1254.
[Abstract] [Full Text] [PDF]


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J Med MicrobiolHome page
Q. V. Tu, M. A. McGuckin, and G. L. Mendz
Campylobacter jejuni response to human mucin MUC2: modulation of colonization and pathogenicity determinants
J. Med. Microbiol., July 1, 2008; 57(7): 795 - 802.
[Abstract] [Full Text] [PDF]


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Hum Mol GenetHome page
D. C. Martin, V. Atmuri, R. J. Hemming, J. Farley, J. S. Mort, S. Byers, S. Hombach-Klonisch, R. Stern, and B. L. Triggs-Raine
A mouse model of human mucopolysaccharidosis IX exhibits osteoarthritis
Hum. Mol. Genet., July 1, 2008; 17(13): 1904 - 1915.
[Abstract] [Full Text] [PDF]


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Plant CellHome page
L. Gutierrez, M. Mauriat, J. Pelloux, C. Bellini, and O. Van Wuytswinkel
Towards a Systematic Validation of References in Real-Time RT-PCR
PLANT CELL, July 1, 2008; 20(7): 1734 - 1735.
[Full Text] [PDF]


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J. Gen. Virol.Home page
L. M. Hedges and K. N. Johnson
Induction of host defence responses by Drosophila C virus
J. Gen. Virol., June 1, 2008; 89(6): 1497 - 1501.
[Abstract] [Full Text] [PDF]


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J. Bacteriol.Home page
C. T. D. Price, A. Bukka, M. Cynamon, and J. E. Graham
Glycine Betaine Uptake by the ProXVWZ ABC Transporter Contributes to the Ability of Mycobacterium tuberculosis To Initiate Growth in Human Macrophages
J. Bacteriol., June 1, 2008; 190(11): 3955 - 3961.
[Abstract] [Full Text] [PDF]


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Mol. Pharmacol.Home page
R. C. Zangar, N. Bollinger, S. Verma, N. J. Karin, and Y. Lu
The Nuclear Factor-{kappa}B Pathway Regulates Cytochrome P450 3A4 Protein Stability
Mol. Pharmacol., June 1, 2008; 73(6): 1652 - 1658.
[Abstract] [Full Text] [PDF]


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Physiol. GenomicsHome page
J. Zagorski, N. Sanapareddy, M. A. Gellar, J. A. Kline, and J. A. Watts
Transcriptional profile of right ventricular tissue during acute pulmonary embolism in rats
Physiol Genomics, June 1, 2008; 34(1): 101 - 111.
[Abstract] [Full Text] [PDF]


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Appl. Environ. Microbiol.Home page
C. W. Tsai, E. A. McGraw, E.-D. Ammar, R. G. Dietzgen, and S. A. Hogenhout
Drosophila melanogaster Mounts a Unique Immune Response to the Rhabdovirus Sigma virus
Appl. Envir. Microbiol., May 15, 2008; 74(10): 3251 - 3256.
[Abstract] [Full Text] [PDF]


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J. Immunol.Home page
E. J. Cornish, B. J. Hurtgen, K. McInnerney, N. L. Burritt, R. M. Taylor, J. N. Jarvis, S. Y. Wang, and J. B. Burritt
Reduced Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Independent Resistance to Aspergillus fumigatus in Alveolar Macrophages
J. Immunol., May 15, 2008; 180(10): 6854 - 6867.
[Abstract] [Full Text] [PDF]



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