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

Reverse transcription (RT) followed by polymerase chain reaction (PCR) is t he technique of choice for analysing mRNA in extremely low abundance. Real- time RT-PCR using SYBR Green I detection combines the ease and necessary ex actness 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 cali bration curve is needed. We have developed, optimised and validated an Insu lin-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) calib ration curve. Above that, the limits, accuracy and variation of these exter nally 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 co nclude that external calibration curve using recDNA is a better model for t he quantification of mRNA than the recRNA calibration model. This model sho wed higher sensitivity, exhibit a larger quantification range, had a higher reproducibility, and is more stable than the recRNA calibration curve.