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

• Moriya Yuka

  Department of Hospital Pharmacy, School of Medicine, Kobe University

• Nakamura Tsutomu

  Department of Hospital Pharmacy, School of Medicine, Kobe University

• Okamura Noboru

  Department of Clinical Evaluation of Pharmacotherapy, Kobe University Graduate School of Medicine

• Sakaeda Toshiyuki

  Department of Hospital Pharmacy, School of Medicine, Kobe University

• Horinouchi Masanori

  Department of Hospital Pharmacy, School of Medicine, Kobe University

• Tamura Takao

  Division of Diabetes, Digestive and Kidney Diseases, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine

• Aoyama Nobuo

  Department of Endoscopy, School of Medicine, Kobe University

• Kasuga Masato

  Division of Diabetes, Digestive and Kidney Diseases, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine

• Okumura Katsuhiko

  Department of Hospital Pharmacy, School of Medicine, Kobe University Department of Clinical Evaluation of Pharmacotherapy, Kobe University Graduate School of Medicine

抄録

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.

収録刊行物

• Ｂｉｏｌｏｇｉｃａｌ　＆　Ｐｈａｒｍａｃｅｕｔｉｃａｌ　Ｂｕｌｌｅｔｉｎ

  Ｂｉｏｌｏｇｉｃａｌ　＆　Ｐｈａｒｍａｃｅｕｔｉｃａｌ　Ｂｕｌｌｅｔｉｎ 29 (3), 535-538, 2006

  公益社団法人 日本薬学会

キーワード

• synthetic DNA template
• authentic cDNA template
• cryopreservation
• real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR)

詳細情報

• CRID: 1390001204624513792
• NII論文ID: 110005602138
• NII書誌ID: AA10885497
• DOI: 10.1248/bpb.29.535
• ISSN: 13475215
• ISSN: 09186158
• NDL書誌ID: 7830447
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I7830447
• Web Site: http://www.jstage.jst.go.jp/article/bpb/29/3/29_3_535/_pdf
• Web Site: https://search.jamas.or.jp/link/ui/2006310944
• 本文言語コード: en
• データソース種別:
  • JaLC
  • NDL
  • Crossref
  • CiNii Articles
• 抄録ライセンスフラグ: 使用不可