Abstract
Real-time polymerase chain reaction (PCR) constitutes a significant improvement over traditional end-point PCR, as it allows the quantification of starting amounts of nucleic acid templates, in real-time. However, quantification requires validation through numerous internal controls and standard curves. We describe in this paper a simple protocol which uses real-time PCR to compare mRNA levels of a gene of interest between different experimental conditions. Comparative real-time PCR can be a relatively low-cost method and does not require sequence-specific fluorescent reporters. Moreover, several genes from a set of experiments can be assessed in a single run. Thus, in addition to providing a comparative profile for the expression of a gene of interest, this method can also provide information regarding the relative abundance of different mRNA species.
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Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (NY) 1993; 11:1026–1030.
Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 1968; 97:77–89.
Pouliot M, Fiset ME, Masse M, Naccache PH, Borgeat P. Adenosine up-regulates cyclooxygenase-2 in human granulocytes: impact on the balance of eicosanoid generation. J Immunol 2002; 169:5279–5286.
Karsai A, Muller S, Platz S, Hauser MT. Evaluation of a homemade SYBR green I reaction mixture for real-time PCR quantification of gene expression. Biotechniques 2002; 32:790–792, 794–796.
Ginzinger DG. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 2002; 30:503–512.
Cadieux JS, Leclerc P, St-Onge M, Dussault AA, Laflamme C, Picard S, Ledent C, Borgeat P, Pouliot M. Potentiation of neutrophil cyclooxygenase-2 by adenosine: an early anti-inflammatory signal. J Cell Sci 2005; 118:1437–1447.
McColl SR, St-Onge M, Dussault AA, Laflamme C, Bouchard L, Boulanger J, Pouliot M. Immunomodulatory impact of the A2A adenosine receptor on the profile of chemokines produced by neutrophils. FASEB J 2006; 20:187–189.
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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Dussault, AA., Pouliot, M. Rapid and simple comparison of messenger rna levels using real-time PCR. Biol. Proced. Online 8, 1–10 (2006). https://doi.org/10.1251/bpo114
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DOI: https://doi.org/10.1251/bpo114