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Biotechnology Letters

, Volume 23, Issue 4, pp 275–282 | Cite as

Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration curves in real-time RT-PCR

  • Michael W. Pfaffl
  • M. Hageleit
Article

Abstract

Reverse transcription (RT) followed by polymerase chain reaction (PCR) is the technique of choice for analysing mRNA in extremely low abundance. Real-time RT-PCR using SYBR Green I detection combines the ease and necessary exactness to be able to produce reliable as well as rapid results. To obtain high accuracy and reliability in RT and real-time PCR a highly defined calibration curve is needed. We have developed, optimised and validated an Insulin-like growth factor-1 (IGF-1) RT-PCR in the LightCycler, based on either a recombinant IGF-1 RNA (recRNA) or a recombinant IGF-1 DNA (recDNA) calibration curve. Above that, the limits, accuracy and variation of these externally standardised quantification systems were determined and compared with a native RT-PCR from liver total RNA. For the evaluation and optimisation of cDNA synthesis rate of recRNA several RNA backgrounds were tested. We conclude that external calibration curve using recDNA is a better model for the quantification of mRNA than the recRNA calibration model. This model showed higher sensitivity, exhibit a larger quantification range, had a higher reproducibility, and is more stable than the recRNA calibration curve.

recombinant DNA recombinant RNA real-time RT-PCR 

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References

  1. Becker-André M,Hahlbrock K (1989) Absolute mRNA quantifi-cation using the polymerase chain reaction (PCR). A novel approach by PCR aided transcript titration assay (PATTY). Nucl. Acids Res. 17: 9437-9446.Google Scholar
  2. Ferré F (1992) Quantitative or semi-quantitative PCR: reality versus Myth. PCR Methods Appl. 2: 1-9.Google Scholar
  3. Förster VT (1948) Zwischenmolekulare Energiewanderung und Fluorescence. Annals of Physics (Leipzig).Google Scholar
  4. Fotsis T,Murphy C,Gannon F (1990) Nucleotide sequence of the bovine insulin like growth factor 1 (IGF-1) and its IGF-1A precursor. Nucl. Acids Res. 18: 676.Google Scholar
  5. Freeman WM,Walker SJ,Vrana KE (1999) Quantitative RT-PCR: pitfalls and potential. BioTechniques 26: 112-22.Google Scholar
  6. Gilliland G,Perrin S,Blanchard K,Bunn HF (1990) Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc. Natl. Acad. Sci. USA 87: 2725-2729.Google Scholar
  7. Lakowicz JR (1983) Energy transfer. In: Principles of Fluorescent Spectroscopy, New York: Plenum Press, pp. 303-339.Google Scholar
  8. Leutenegger CM,Mislin CN,Sigrist B,Ehrengruber MU,Hofmann-Lehmann R,Lutz H (1999) Quantitative real-time PCR for the measurement of feline cytokine mRNA. Vet. Immunol. Immunopathol. 71: 291-305Google Scholar
  9. Loeffler J,Henke N,Hebart H,Schmidt D,Hagmeyer L,Schumacher U,Hermann E (2000) Quantification of fungal DNA by using fluorescence resonance energy transfer and the LightCycler system. J. Clin. Microbiol. 38: 586-590.Google Scholar
  10. McPherson MJ,Quirke P,Taylor GR (1991) PCR, A Practical Approach. Oxford: Oxford University Press.Google Scholar
  11. Morrison T,Weis JJ,Wittwer CT (1998) Quantification of lowcopy transcripts by continuous SYBR Green I monitoring during amplification. BioTechniques 24: 954-962.Google Scholar
  12. Pfaffl MW (2000) Development and validation of an externally standardised quantitative Insulin like growth factor-1 (IGF-1) RT-PCR using LightCycler SYBRr Green I technology. In: Meuer S,Wittwer C,Nakagawara K, eds. Rapid Cycle Real-Time PCR, Methods and Applications. Heidelberg: Springer-Verlag Press (in press).Google Scholar
  13. Pfaffl MW,Elsasser F,Meyer HHD,Sauerwein H (1998a). Exogenous porcine growth hormone (GH) up-regulates hepatic and muscle insulin-like growth factor-1 (IGF-1) mRNA expression in two different genotypes of pigs. Exp. Clin. Endocrinol. Diabetes 106: 50.Google Scholar
  14. Pfaffl MW,Meyer HHD,Sauerwein H (1998b) Quantification of the insulin like growth factor-1 (IGF-1) mRNA: development and validation of an internally standardised competitive reverse transcription-polymerase chain reaction. Exp. Clin. Endocrinol. Diabetes 106: 502-512.Google Scholar
  15. Siebert PD,Larrick JW (1992) Competitive PCR. Nature 359: 557-558.Google Scholar
  16. Simmen FA (1991) Expression of the insulin-like growth factor-I gene and its products: complex regulation by tissue specific and hormonal factors. Domest. Anim. Endocrinol. 8: 165-178.Google Scholar
  17. Souazé F,Ntodou-Thomé A,Tran CY,Rostene W,Forgez P (1996) Quantitative RT-PCR: limits and accuracy. BioTechniques 21: 280-285.Google Scholar
  18. Thissen JP,Ketelslegers JM,Underwood LE (1994) Nutritional regulation of the insulin-like growth factors. Endocr. Rev. 15: 80-101.Google Scholar
  19. Winer J,Jung CK,Shackel I,Williams PM (1999) Development and validation of real-time quantitative reverse transcriptasepolymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal. Biochem. 270: 41-49.Google Scholar
  20. Wittwer CT,Garling DJ (1991) Rapid cycle DNA amplification: time and temperature optimization. BioTechniques 10: 76-83.Google Scholar
  21. Wittwer CT,Ririe KM,Andrew RV,David DA,Gundry RA,Balis UJ (1997) The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. BioTechniques 22: 176-181.Google Scholar
  22. Zimmermann K,Mannhalter JW (1996) Technical aspects of quantitative competitive PCR. BioTechniques 21: 268-279.Google Scholar

Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • Michael W. Pfaffl
    • 1
  • M. Hageleit
    • 1
  1. 1.Institute of Physiology, FML-Weihenstephan, Center of Life and Food SciencesTechnical University of MünichGermany

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