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


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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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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