文章基本信息

标题：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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作者：Yuka Moriya ; Tsutomu Nakamura ; Noboru Okamura 等
期刊名称：Biological and Pharmaceutical Bulletin
印刷版ISSN：0918-6158
电子版ISSN：1347-5215
出版年度：2006
卷号：29
期号：3
页码：535-538
DOI：10.1248/bpb.29.535
出版社：The Pharmaceutical Society of Japan
摘要：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 °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.
关键词：synthetic DNA template;authentic cDNA template;cryopreservation;real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR)