Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction.
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 10(8)-10(3) 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 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.
Johnson
M R
MR
Department of Pharmacology and Toxicology and Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Wang
K
K
Smith
J B
JB
Heslin
M J
MJ
Diasio
R B
RB
D042943:Dihydrouracil Dehydrogenase (NADP); D006801:Humans; D015345:Oligonucleotide Probes; D010088:Oxidoreductases / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors
D015345:Oligonucleotide Probes; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP)