Rapid quantitative PCR determination of relative gene expression in tumor specimens using high-pressure liquid chromatography.

A reverse transcriptase polymerase chain reaction (rt-PCR) for quantification of gene expression has been optimized for analysis of folylpolyglutamate synthase (FPGS) and thymidylate synthase (TS), using beta-actin as an internal standard (house-keeping gene). Total RNA was isolated from tumor tissue, reversely transcribed to cDNA and PCR amplified with primers specific for TS, FPGS and beta-actin in separate vials. PCR products were separated and quantified by high-pressure liquid chromatography (HPLC) without addition of radioactive or fluorescent markers, which minimizes labor and occupational hazards. The day-to-day variation in the HPLC analysis was 2.7% and the within sample variations for rt-PCR/HPLC analysis of TS and FPGS were 18.5% for both assays. This method provides a tool for convenient gene expression analysis in clinical biopsies.