Comparison of Synthetic DNA Templates With Authentic cDNA Templates in Terms of Quantification by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction

Synthetic DNA templates were compared with authentic CDNA templates as standards for the real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The single-stranded DNA template used here targeted the multidrug resistant transporter P-glycoprotein/MDR1. The double-stranded DNA template, targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was synthesized using an exonuclease-free large fragment E. coli DNA polymerase I. The human colon carcinoma cell line Caco-2 and human duodenum biopsies were used to prepare the authentic CDNA templates. The standard lines were comparable for the synthetic DNA templates and authentic CDNA templates. Long-term cryopreservation at -80℃ resulted in the destabilization of the synthetic single-stranded DNA template compared with the authentic CDNA templates in the case of MDRl, whereas for GAPDH, the stability of the synthetic double-stranded DNA template was comparable with that of the authentic CDNA ternplates. Even for the synthetic DNA templates, repetitive freeze-thawing resulted in destabilization, especially at lower concentrations, and degradation products might have interfered with the RT-PCR'S efficiency. The synthetic DNA templates are better than the authentic CDNA templates, but more than 5 cycles of repetitive freeze-thawing should be avoided.