Four quantitative reverse transcription-PCR (RT-PCR) methods were compared to evaluate the time course of mRNA formation and decay. Mouse fibroblasts (NIH 3T3) transfected with the human -globin open reading frame/c-myc 3-untranslated region chimeric gene under control of the c-fos promoter (fosglo-myc) were used for serum-inducible transcription. The amount of fos-glo-myc mRNA, relative to -actin, was measured by quantitative, RT-PCR at various times following the addition of serum to serumstarved fibroblasts transfected with the chimeric gene. Both endpoint (band densitometry and probe hybridization) and real-time (SYBR green and Taq-Man) PCR methods were used to assay the identical cDNA.
The real-time methods produced a 4-to 5-log dynamic range of amplification, while the dynamic range of the endpoint assays was 1-log. The real-time and probe hybridization assays produced a comparable level of sensitivity that was considerably greater than band densitometry. The coefficient of variation from 22 replicate PCR reactions was 14.2 and 24.0% for the SYBR green and TaqMan detection, respectively, and 44.9 and 45.1% for the band densitometry and probe hybridization assays, respectively. The rank order for the values of r 2 obtained from the linear regression of the first-order mRNA decay plots was SYBR green > TaqMan > probe hybridization > band densitometry. Real-time PCR is more precise and displays a greater dynamic range than endpoint PCR. Among the real-time methods, SYBR green and TaqMan assays produced comparable dynamic range and sensitivity while SYBR green detection was more precise and produced a more linear decay plot than TaqMan detection.