Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction

Martin R. Johnson, Kangsheng Wang, Jeffrey B. Smith, Martin J. Heslin, Robert B. Diasio

Research output: Contribution to journalArticle

315 Scopus citations

Abstract

Several recent studies have reported a correlation between intratumor dihydropyrimidine dehydrogenase (DPD) messenger RNA (mRNA) levels and sensitivity to 5-fluorouracil (5-FU). However, significant tissue requirements and labor-intensive methodology have limited the large-scale studies necessary for statistical validation. In addition, the semiquantitative results obtained by these methods further limit their application. We have developed a realtime reverse transcription-PCR (RT-PCR) assay, based on TaqMan fluorescence methodology, capable of rapid and accurate quantitation of DPD mRNA levels in biopsy-sized tissue samples. Results obtained with this approach indicate a linear dynamic range of 108- 103 DPD mRNA copies, with an intraassay variation of <5%. We evaluated the data using three different methods (absolute standard curve, relative standard curve, and comparative C(T)) and show them to be equivalent. This RT-PCR assay was validated by quantitative comparison to Northern blot analysis in five tissues. In addition, analysis of 18 colorectal tumor and liver tissue specimens demonstrated a significant correlation (r2 = 0.90) between DPD enzyme activity and mRNA levels. This method provides the first high-throughput, reproducible, and sensitive technique capable of determining DPD mRNA expression levels in nanogram amounts of total RNA. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)175-184
Number of pages10
JournalAnalytical Biochemistry
Volume278
Issue number2
DOIs
StatePublished - Feb 15 2000

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Keywords

  • Dihydropyrimidine dehydrogenase
  • Pharmacogenetics
  • Pharmacogenomics
  • Quantitative RT-PCR
  • Real-time PCR
  • TaqMan

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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