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

✍ Scribed by Martin R. Johnson; Kangsheng Wang; Jeffrey B. Smith; Martin J. Heslin; Robert B. Diasio

• Publisher: Elsevier Science
• Year: 2000
• Tongue: English
• Weight: 154 KB
• Volume: 278
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1999.4461

✦ Synopsis

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 10 8 -10 3 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 (r 2 ‫؍‬ 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.