Quantitation of Dihydropyrimidine Dehydrogenase Expression by Real-Time Reverse Transcription Polymerase Chain Reaction

• Published in: Analytical biochemistry Vol. 278; no. 2; pp. 175 - 184
• Main Authors: Johnson, Martin R., Wang, Kangsheng, Smith, Jeffrey B., Heslin, Martin J., Diasio, Robert B.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.02.2000
• Subjects: dihydropyrimidine dehydrogenase; Dihydrouracil Dehydrogenase (NADP); Humans; Oligonucleotide Probes; Oxidoreductases - analysis; Oxidoreductases - biosynthesis; Oxidoreductases - genetics; pharmacogenetics; pharmacogenomics; Polymerase Chain Reaction - methods; quantitative RT-PCR; real-time PCR; TaqMan; Time Factors; dihydropyrimidine dehydrogenase; pharmacogenomics; real-time PCR; TaqMan; pharmacogenetics; quantitative RT-PCR
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1999.4461

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 real-time 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 intra-assay variation of <5%. We evaluated the data using three different methods (absolute standard curve, relative standard curve, and comparative CT) 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.