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 quantif ication of gene expression has been optimized for analysis of folylpol yglutamate synthase (FPGS) and thymidylate synthase (TS), using beta-a ctin as an internal standard (house-keeping gene). Total RNA was isola ted 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 chr omatography (HPLC) without addition of radioactive or fluorescent mark ers, which minimizes labor and occupational hazards. The day-to-day va riation 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. T his method provides a tool for convenient gene expression analysis in clinical biopsies.