Simultaneous measurement of multiple mRNAs with a single control by quantitative competitive reverse...

Theoretical considerations for extending the application of quantitative competitive polymerase chain reaction (qc-PCR) to include the simultaneous measurement of multiple mRNAs, specifically the mammalian glucose transporters Glut1 and Glut4, are presented with experimental data in which the accuracy and flexibility of the system are examined. This method reliably measures changes in the initial concentration for each of three target DNA sequences. The reaction is not acutely sensitive to variations in either the primer sites or internal sequence, and although the initial concentrations of the target DNAs did affect the relative amplification efficiencies, the effect was limited and did not prohibit quantification. This PCR system was able to reliably detect differences as little as 50% in the initial concentration of the Glut1 target DNA sequence. Therefore, with the appropriate controls, PCR can be extended to include the simultaneous quantification of more than one target DNA with a single internal control.