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 degrees C resulted in the destabilization of the synthetic single-stranded DNA template compared with the authentic cDNA templates in the case of MDR1, whereas for GAPDH, the stability of the synthetic double-stranded DNA template was comparable with that of the authentic cDNA templates. 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.
Moriya
Yuka
Y
Department of Hospital Pharmacy, School of Medicine, Kobe University, Japan.
Nakamura
Tsutomu
T
Okamura
Noboru
N
Sakaeda
Toshiyuki
T
Horinouchi
Masanori
M
Tamura
Takao
T
Aoyama
Nobuo
N
Kasuga
Masato
M
Okumura
Katsuhiko
K
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018938:Caco-2 Cells; D015925:Cryopreservation; D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry*; D018076:DNA, Complementary / Q000737:chemistry*; D004273:DNA, Neoplasm / Q000737:chemistry; D003627:Data Interpretation, Statistical; D004386:Duodenum / Q000737:chemistry; D005615:Freezing; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008297:Male; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013698:Templates, Genetic
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018076:DNA, Complementary; D004273:DNA, Neoplasm; D009841:Oligonucleotides; D004247:DNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases