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Quantification of thymidylate synthase gene expression in human gastrointestinal carcinoma tissues using competitive
K Omura1, M Morishita, K Kawakami
1Department of Surgery 1, Kanazawa University School of Medicine Faculty of Medicine, Japan. omura@med.kanazawa-u.ac.jp
Background/Aims:
Thymidylate synthase (TS) is a target enzyme for 5-fluorouracil (5-FU). It is important to know the TS expression level in tumor tissue for chemotherapy. We evaluated the TS expression level using competitive polymerase chain reaction (cPCR), and estimated the reliability and reproducibility of this method.
Methodology:
TS expression was assessed by both Fluorodeoxyuridine-monophosphate (FdUMP) binding assay and cPCR in 9 human adenocarcinoma cell lines and 50 human adenocarcinoma tissues obtained by surgical resection. TS expression was also assessed by cPCR in nine biopsy specimens obtained before surgery. We synthesized TS and beta-actin competitors. Following cPCR, PCR products were quantified on ethidium bromide-stained gels using a digital image analyzer. TS mRNA/beta-actin mRNA ratio was used to determine relative TS expression level.
Results:
The number of FdUMP binding sites on TS and TS mRNA/beta-actin mRNA ratio were significantly correlated in both cell lines and surgically resected specimens (p < 0.01). A linear regression line formed from the data points was obtained for TS mRNA/beta-actin mRNA ratios in surgical specimens versus TS mRNA/beta-actin mRNA ratios in biopsy specimens (p < 0.01).
Conclusions:
Assessment of TS expression by cPCR using our competitors was accurate and reproducible.