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Analyzing real-time PCR data by the comparative CT method


Two different methods of presenting quantitative gene expression exist: absolute and relative quantification. Absolute quantification calculates the copy number of the gene usually by relating the PCR signal to a standard curve. Relative gene expression presents the data of the gene of interest relative to some calibrator or internal control gene. A widely used method to present relative gene expression is the comparative CT method also referred to as the 2 - ΔΔ C T method. This protocol provides an overview of the comparative CT method for quantitative gene expression studies. Also presented here are various examples to present quantitative gene expression data using this method.

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Figure 1: Real-time PCR output, calculation of CT.
Figure 2: Real-time PCR plots from gene expression profiling.
Figure 3: Real-time PCR expression profiling of microRNA in pancreas tissue.

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  1. Kim, Y.W. et al. Anti-inflammatory effects of liquiritigenin as a consequence of the inhibition of NF-kappaB-dependent iNOS and proinflammatory cytokines production. Br. J. Pharmacol. 154, 165–173 (2008).

    Article  CAS  Google Scholar 

  2. Pal, S. et al. Low levels of miR-92b/96 induce PRMT5 translation and H3R8/H4R3 methylation in mantle cell lymphoma. EMBO J. 26, 3558–3569 (2007).

    Article  CAS  Google Scholar 

  3. Ren, Z. et al. IGFBP3 mRNA expression in benign and malignant breast tumors. Breast Cancer Res. 9, R2 (2007).

    Article  Google Scholar 

  4. Higashibata, A. et al. Decreased expression of myogenic transcription factors and myosin heavy chains in Caenorhabditis elegans muscles developed during spaceflight. J. Exp. Biol. 209, 3209–3218 (2006).

    Article  CAS  Google Scholar 

  5. Woods, D.C., Alvarez, C. & Johnson, A.L. Cisplatin-mediated sensitivity to TRAIL-induced cell death in human granulosa tumor cells. Gynecol. Oncol. 108, 632–640 (2008).

    Article  CAS  Google Scholar 

  6. Calin, G.A. et al. Ultraconserved regions encoding ncRNAs are altered in human leukemias and carcinomas. Cancer Cell 12, 215–229 (2007).

    Article  CAS  Google Scholar 

  7. Zhao, M. et al. Lipofectamine RNAiMAX: an efficient siRNA transfection reagent in human embryonic stem cells. Mol. Biotechnol. (in the press).

  8. Spänkuch, B. et al. Downregulation of Plk1 expression by receptor-mediated uptake of antisense oligonucleotide-loaded nanoparticles. Neoplasia 10, 223–234 (2008).

    Article  Google Scholar 

  9. Paik, S. et al. A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N. Engl. J. Med. 351, 2817–2826 (2004).

    Article  CAS  Google Scholar 

  10. Chen, C. et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 33, e179 (2005).

    Article  Google Scholar 

  11. Niesters, H.G. Quantitation of viral load using real-time amplification techniques. Methods 25, 419–429 (2001).

    Article  CAS  Google Scholar 

  12. Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45 (2001).

    Article  CAS  Google Scholar 

  13. Liu, W. & Saint, D.A. A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal. Biochem. 302, 52–59 (2002).

    Article  CAS  Google Scholar 

  14. Rutledge, R.G. Sigmoidal curve-fitting redefines quantitative real-time PCR with the prospective of developing automated high-throughput applications. Nucleic Acids Res. 32, e178 (2004).

    Article  CAS  Google Scholar 

  15. Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408 (2001).

    Article  CAS  Google Scholar 

  16. Swillens, S., Dessars, B. & Housni, H.E. Revisiting the sigmoidal curve fitting applied to quantitative real-time PCR data. Anal. Biochem. 373, 370–376 (2008).

    Article  CAS  Google Scholar 

  17. Griffiths-Jones, S. The microRNA Registry. Nucleic Acids Res. 32, D109–D111 (2004).

    Article  CAS  Google Scholar 

  18. Eisen, M.B., Spellman, P.T., Brown, P.O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95, 14863–14868 (1998).

    Article  CAS  Google Scholar 

  19. Sun, S. et al. TINY, a dehydration-responsive element (DRE)-binding protein-like transcription factor connecting the DRE- and ethylene-responsive element-mediated signaling pathways in Arabidopsis. J. Biol. Chem. 283, 6261–6271 (2008).

    Article  CAS  Google Scholar 

  20. Tan, K.P., Yang, M. & Ito, S. Activation of nuclear factor (erythroid-2 like) factor 2 by toxic bile acids provokes adaptive defense responses to enhance cell survival at the emergence of oxidative stress. Mol. Pharmacol. 72, 1380–1390 (2007).

    Article  CAS  Google Scholar 

  21. O'Rourke, J.P. & Ness, S.A. Alternative RNA splicing produces multiple forms of c-Myb with unique transcriptional activities. Mol. Cell. Biol. 28, 2091–2101 (2008).

    Article  CAS  Google Scholar 

  22. Schmittgen, T.D. & Zakrajsek, B.A. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J. Biochem. Biophys. Methods 46, 69–81 (2000).

    Article  CAS  Google Scholar 

  23. Mygind, T. et al. Determination of PCR efficiency in chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae. BMC Microbiol. 2, 17 (2002).

    Article  Google Scholar 

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Correspondence to Thomas D Schmittgen.

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Schmittgen, T., Livak, K. Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3, 1101–1108 (2008).

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