Validation of real-time reverse-transcription-polymerase chain reaction for quantification of...

Capecitabine is an oral fluoropyrimidine carbamate activated sequentially in both liver and tumor tissues by carboxylesterases, cytidine deaminase, and thymidine phosphorylase. 5-Fluorouracil is inactivated by dihydropyrimidine dehydrogenase and targets thymidylate synthase. Here we report the validation of the real-time polymerase chain reaction assay for the quantification of the transcripts of the different enzymes involved in capecitabine activation. The method is specific, sensitive, and linear over 2-3 logs of RNA input. It is reproducible with less than 5% intraday variability and less than 10% interday variability. Five reference genes were tested for normalization. POLR2A was selected since it reduced variability between samples, demonstrated levels of expression similar to those of the genes of interest, and its expression was not modified by capecitabine treatment in samples from preclinical studies. The method was robust as the gene expression profiles from six colon cancer cell lines obtained by this method were similar to microarray data. Finally, the method was able to detect changes in gene expression in xenograft tumors treated with capecitabine. It could therefore constitute the method of choice for future correlative studies in patients receiving capecitabine.