Comparison of synthetic DNA templates with authentic cDNA templates in terms of quantification by real-time quantitative reverse transcription polymerase chain reaction
Comparison of synthetic DNA templates with authentic cDNA templates in terms of quantification by real-time quantitative reverse transcription polymerase chain reaction
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DOI：
10.1248/bpb.29.535
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发表时间：
2006-03-01
影响因子：
2
通讯作者：
Okumura, K
中科院分区：
文献类型：
--
作者：
Moriya, Y;Nakamura, T;Okumura, K
将合成的DNA模板与真品的DNA模板进行比较，作为实时定量逆转录聚合酶链式反应(RT-PCR)的标准。这里使用的单链DNA模板针对多药耐药转运蛋白P-糖蛋白/mdr1。用无外切酶的大片段大肠杆菌DNA聚合酶I合成了针对3-磷酸甘油醛脱氢酶(GAPDH)的双链DNA模板，并以人结肠癌细胞系Caco-2和人十二指肠活检组织为模板。标准品系与人工合成的DNA模板和正品的DNA模板具有可比性。在MDR1的情况下，长期冷冻保存导致合成的单链DNA模板与真实的cDNA模板相比不稳定，而对于GAPDH，合成的双链DNA模板的稳定性与真实的cDNA模板的稳定性相当。即使对于合成的DNA模板，重复冻融也会导致不稳定，特别是在较低浓度下，降解产物可能会干扰RT-PCR的效率。人工合成的DNA模板优于真品DNA模板，但应避免5个以上的重复冻融循环。
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.