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
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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 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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 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. 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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 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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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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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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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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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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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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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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 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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 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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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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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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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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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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 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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 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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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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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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