Comparison of synthetic DNA templates with authentic cDNA templates in terms of quantification by real-time quantitative reverse transcription polymerase chain reaction

Biological & Pharmaceutical Bulletin
Yuka MoriyaKatsuhiko Okumura

Abstract

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...Continue Reading

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Citations

Apr 2, 2009·Biological & Pharmaceutical Bulletin·Kazuya IshidaYukiya Hashimoto
Mar 26, 2009·Journal of Microbiological Methods·Ambroos StalsEls Van Coillie
May 12, 2009·European Journal of Pharmaceutical Sciences : Official Journal of the European Federation for Pharmaceutical Sciences·Daisuke YamasakiToshiyuki Sakaeda
Sep 3, 2009·Biopharmaceutics & Drug Disposition·Shiro FukumoriYukiya Hashimoto
Aug 20, 2008·Journal of Neuroscience Research·Naomi L CookCorinna van den Heuvel
May 14, 2011·Biopharmaceutics & Drug Disposition·Asuka HorieYukiya Hashimoto
Sep 27, 2014·Biopharmaceutics & Drug Disposition·Asuka HorieYukiya Hashimoto
Jun 5, 2016·Virology Journal·Karissa A LemireMichael T McIntosh
Mar 3, 2012·PloS One·Helena StorchovaMatthew S Olson
Sep 25, 2008·Analytical Biochemistry·Stefan Cikos, Juraj Koppel
Feb 1, 2007·Journal of Animal Science·M M VickB P Fitzgerald

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