Skip to main content
SpringerLink
Log in

Real-time PCR: A review of approaches to data analysis

  • Published:
Applied Biochemistry and Microbiology Aims and scope Submit manuscript

Abstract

The registration of the accumulation of polymerase chain reaction (PCR) products in the course of amplification (real-time PCR) requires specific equipment, i.e., detecting amplifiers capable of recording the level of fluorescence in the reaction tube during amplicon formation. When the time of the reaction is complete, researchers are able to obtain DNA accumulation graphs. This review discusses the most promising algorithms of the analysis of real-time PCR curves and possible errors, caused by the software used or by operators’ mistakes. The data included will assist researchers in understanding the features of a method to obtain more reliable results.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Walker, N.J., Science, 2002, vol. 296, no. 5567, pp. 557–559.

    Article  PubMed  CAS  Google Scholar 

  2. Lay, M.J. and Wittwer, C.T., Clin. Chem., 1997, vol. 43, no. 12, pp. 2262–2267.

    PubMed  CAS  Google Scholar 

  3. Aslanidis, C., Nauck, M., and Schmitz, G., BioTechniques, 1999, vol. 27, no. 2, pp. 234–238.

    PubMed  CAS  Google Scholar 

  4. Bustin, S.A., J. Mol. Endocrinol, 2000, vol. 25, no. 2, pp. 169–193.

    Article  PubMed  CAS  Google Scholar 

  5. Schmittgen, T.D., Methods, 2001, vol. 25, no. 4, pp. 383–385.

    Article  PubMed  CAS  Google Scholar 

  6. Giulietti, A., Overbergh, L., Valckx, D., Decallonne, B., Bouillon, R., and Mathieu, C., Methods, 2001, vol. 25, no. 4, pp. 386–401.

    Article  PubMed  CAS  Google Scholar 

  7. Feinberg, M., Fernandez, S., Cassard, S., and Bertheau, Y., J. AOAC Int., 2005, vol. 88, no. 2, pp. 558–573.

    PubMed  CAS  Google Scholar 

  8. Holland, P.M., Abramson, R.D., Watson, R., and Gelfand, D.H., Proc. Natl. Acad. Sci. USA, 1991, vol. 88, no. 16, pp. 7276–7280.

    Article  PubMed  CAS  Google Scholar 

  9. Tyagi, S. and Kramer, F.R., Nat. Biotechnol, 1996, vol. 14, no. 3, pp. 303–308.

    Article  PubMed  CAS  Google Scholar 

  10. Morrison, T.B., Weis, J.J., and Wittwer, C.T., BioTechniques, 1998, vol. 24, no. 6, pp. 954–958.

    PubMed  CAS  Google Scholar 

  11. Winn-Deen, E.S., Mol. Diagn, 1998, vol. 3, no. 4, pp. 217–222.

    Article  PubMed  CAS  Google Scholar 

  12. Thelwell, N., Millington, S., Solinas, A., Booth, J., and Brown, T., Nucleic Acid Res., 2000, vol. 28, no. 19, pp. 3752–3761.

    Article  PubMed  CAS  Google Scholar 

  13. Livak, K.J. and Schmittgen, T.D., Methods, 2001, vol. 25, no. 4, pp. 402–408.

    Article  PubMed  CAS  Google Scholar 

  14. Liu, W. and Saint, D.A., Anal. Biochem., 2002, vol. 302, no. 1, pp. 52–59.

    Article  PubMed  CAS  Google Scholar 

  15. Bar, T., Stahlberg, A., Muszta, A., and Kubista, M., Nucleic Acid Res., 2003, vol. 31, no. 17.

  16. Pfaffl, M.W., Nucleic Acid Res., 2001, vol. 29, no. 9.

  17. Teo, I.A., Choi, J.W., Morlese, J., Taylor, G., and Shaunak, S., J. Immunol. Methods, 2002, vol. 270, no. 1, pp. 119–133.

    Article  PubMed  CAS  Google Scholar 

  18. Ball, T.B. and Plummer, F.A., HayGlass K.T., Int. Arch. Allergy Immunol, 2003, vol. 130, no. 1, pp. 82–86.

    Article  PubMed  CAS  Google Scholar 

  19. Bolufer, P., Colomer, D., Gomez, M.T., Martinez, J., Gonzalez, S.M., Gonzalez, M., Nomdedeu, J., Bellosillo, B., Barragan, E., Lo-Coco, F., Diverio, D., Hermosin, L., Garcia-Marco, J., DeJuan, M.D., Barros, F., Romero, R., and Sanz, M.A., Clin. Chem., 2004, vol. 50, no. 6, pp. 1088–1092.

    Article  PubMed  CAS  Google Scholar 

  20. Burkardt, H.J., Clin. Chem. Lab. Med, 2000, vol. 38, no. 2, pp. 87–91.

    Article  PubMed  CAS  Google Scholar 

  21. Peters, I.R., Helps, C.R., Hall, E.J., and Day, M.J., J. Immunol. Methods, 2004, vol. 286, nos. 1–2, pp. 203–217.

    Article  PubMed  CAS  Google Scholar 

  22. Wilhelm, J., Pingoud, A., and Hahn, M., Anal. Biochem., 2003, vol. 317, no. 2, pp. 218–225.

    Article  PubMed  CAS  Google Scholar 

  23. Muller, P.Y., Janovjak, H., Miserez, A.R., and Dobbie, Z., BioTechniques, 2002, vol. 32, no. 6, pp. 1372–1379.

    PubMed  CAS  Google Scholar 

  24. Peirson, S.N., Butler, J.N., and Foster, R.G., Nucleic Acid Res., 2003, vol. 31, no. 14.

  25. Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Erlich, H., Cold Spring Harb. Symp. Quant. Biol, 1986, vol. 51, no. 1, pp. 263–273.

    PubMed  CAS  Google Scholar 

  26. Nogva, H.K. and Rudi, K., BioTechniques, 2004, vol. 37, pp. 246–253.

    PubMed  CAS  Google Scholar 

  27. Bustin, S.A., in A-Z of Quantitative PCR, Tsigelny, I.S., Ed., La Jolla (USA): Int. Univ. Line, 2003, p. 7.

    Google Scholar 

  28. Rutledge, R.G. and Cote, C., Nucleic Acid Res., 2003, vol. 31, no. 16.

  29. Arezi, B., Xing, W., Sorge, J.A., and Hogrefe, H.H., Anal. Biochem., 2003, vol. 321, no. 2, pp. 226–235.

    Article  PubMed  CAS  Google Scholar 

  30. Sikorsky, J.A., Primerano, D.A., Fenger, T.W., and Denvir, J., Biochem. Biophys. Res. Commun., 2004, vol. 323, no. 3, pp. 823–830.

    Article  PubMed  CAS  Google Scholar 

  31. Hunt, M., Real Time PCR. Microbiology and Immunology On-Line, Columbia, (USA): Univ. South Carolina, 2005, pp. 1–15.

    Google Scholar 

  32. Liu, W. and Saint, D.A., Biochem. Biophys. Res. Commun., 2002, vol. 294, no. 2, pp. 347–353.

    Article  PubMed  CAS  Google Scholar 

  33. Lukyanov, K.A., Matz, M.V., Bogdanova, E.A., Gurskaya, N.G., and Lukyanov, S.A., Nucleic Acid Res., 1996, vol. 24, no. 11, pp. 2194–2195.

    Article  PubMed  CAS  Google Scholar 

  34. Liss, B., Nucleic Acid Res., 2002, vol. 30, no. 17, p. e89.

    Article  PubMed  Google Scholar 

  35. Hartshorn, C., Anshelevich, A., and Wangh, L.J., BMC Biotechnol, 2005, vol. 5, no. 1, p. 2.

    Article  PubMed  Google Scholar 

  36. Pfaffl, M.W., in Rapid Cycle Real-time PCR, Methods and Applications, Meuer, S., Wittwer, C., and Nakagawara, K., Eds., Heidelberg: Springer Press, 2001, pp. 1–12.

    Google Scholar 

  37. Ramakers, C., Ruijter, J.M., Deprez, R.H., and Moorman, A.F., Neurosci. Lett., 2003, vol. 339, no. 1, pp. 62–66.

    Article  PubMed  CAS  Google Scholar 

  38. Wilhelm, J. and Pingoud, A., Chembiochem., 2003, vol. 4, no. 11, pp. 1120–1128.

    Article  PubMed  CAS  Google Scholar 

  39. Rasmussen, R., in Rapid Cycle Real-time PCR, Methods and Applications, Meuer, S., Wittwer, C., and Nakagawara, K., Eds., Heidelberg: Springer Press, 2001, pp. 21–34.

    Google Scholar 

  40. Stahlberg, A., Aman, P., Ridell, B., Mostad, P., and Kubista, M., Clin. Chem., 2003, vol. 49, no. 1, pp. 51–59.

    Article  PubMed  CAS  Google Scholar 

  41. Peccoud, J. and Jacob, C., in Statistical Estimations of PCR Amplification Rates. Gene Quantification, Ferre, F., Ed., New York: Birkhauser, 1998, pp. 111–128.

    Google Scholar 

  42. Shiao, Y.H., BMC Biotechnol., 2003, vol. 3, no. 12, p. 22.

    Article  PubMed  Google Scholar 

  43. Tichopad, A., Dzidic, A., and Pfaffl, M.W., Biotechnol. Lett., 2002, vol. 24, no. 4, pp. 2053–2056.

    Article  CAS  Google Scholar 

  44. Tichopad, A., Dilger, M., Schwarz, G., and Pfaffl, M.W., Nucleic Aids Res, 2003, vol. 31, no. 20, p. e122.

    Article  Google Scholar 

  45. Rutledge, R.G., Nucleic Acid Res., 2004, vol. 32, no. 22, p. e178.

    Article  PubMed  CAS  Google Scholar 

  46. Zhao, S. and Fernald, R.D., J. Comput. Biol, 2005, vol. 12, no. 8, pp. 1047–1064.

    Article  PubMed  CAS  Google Scholar 

  47. Schoder, D., Schmalwieser, A., Schauberger, G., Kuhn, M., Hoorfar, J., and Wagner, M., Clin. Chem., 2003, vol. 49, no. 6, pp. 960–963.

    Article  PubMed  CAS  Google Scholar 

  48. Schoder, D., Schmalwieser, A., Schauberger, G., Hoorfar, J., Kuhn, M., and Wagner, M., J. Clin. Microbiol., 2005, vol. 43, no. 6, pp. 2724–2728.

    Article  PubMed  CAS  Google Scholar 

  49. Larionov, A., Krause, A., and Miller, W., BMC Bioinformatics, 2005, vol. 6, no. 1, p. 62.

    Article  PubMed  Google Scholar 

  50. US Patent no. 20 050 227 257, 2005.

  51. Cichocki, L., Hecking, C., Bareza, N., Gasser, S., Pilger, E., Klein, W., and Gasser, R., J. Clin. Basic Cardiol, 2004, vol. 7, nos. 1–4, pp. 26–27.

    CAS  Google Scholar 

  52. Pfaffl, M.W., Horgan, G.W., and Dempfle, L., Nucleic Acid Res., 2002, vol. 30, no. 9, p. e36.

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. DNA-Technology JSC, Moscow, 115478, Russia

    D. V. Rebrikov & D. Yu. Trofimov

Authors
  1. D. V. Rebrikov
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D. Yu. Trofimov
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Original Russian Text © D.V. Rebrikov, D.Yu. Trofimov, 2006, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2006, Vol. 42, No. 5, pp. 520–528.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rebrikov, D.V., Trofimov, D.Y. Real-time PCR: A review of approaches to data analysis. Appl Biochem Microbiol 42, 455–463 (2006). https://doi.org/10.1134/S0003683806050024

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0003683806050024

Keywords

Search

Navigation