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  • 标题:Comparison of Synthetic DNA Templates with Authentic cDNA Templates in Terms of Quantification by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction
  • 本地全文:下载
  • 作者:Yuka Moriya ; Tsutomu Nakamura ; Noboru Okamura
  • 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)
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