QUANTITATIVE REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION FOR PROSTATE-SPECIFIC ANTIGEN MESSENGER-RNA

Objective: To develop a quantitative reverse transcriptase-polymerase chain reaction assay for monitoring the prostate-specific antigen (PSA ) mRNA. Methods: PSA mRNA is amplified, in parallel, with the mRNA of beta-actin, a housekeeping gene. The ratio of the amplification produc ts obtained reflects the relative amount of PSA mRNA with respect to a ctin mRNA. During PCR, digoxigenin-dUTP is incorporated in the amplifi ed sequences. The PCR products are analyzed separately by time-resolve d immunofluorometric hybridization assays, using specific probes immob ilized in microtiter wells. The hybrids are reacted with alkaline phos phatase-labeled anti-digoxigenin antibody. The phosphate ester of fluo rosalicylate is used as a substrate. The fluorosalicylate produced for ms a fluorescent complex with Tb3+-EDTA which is measured by time-reso lved fluorometry. Results: The hybridization assays for both PSA and a ctin amplification products show linearity in the range of 1.4-110 pmo l/L. The exponential phase of PCR amplification extends up to 200,000 and 100,000 PSA and actin cDNA molecules, respectively. We prepared mi xtures containing various numbers of LNCaP cells in one million cells that do not express PSA and used them as samples in the proposed assay . The ratio of the fluorescence values obtained after analysis of PSA and actin amplification products is linearly related to the number of LNCaP cells in the range of 20 to 3000 cells. Reproducibility studies demonstrate %CVs for the fluorescence ratios of 14.7, 11.8, and 12.2 w hen samples containing 150, 300 and 1600 LNCaP cells were analyzed (n = 4). Conclusions: A quantitative analytical methodology is provided f or monitoring PSA mRNA. The assay is expected to be beneficial in the study of prostate cancer spread.