Quantitative reverse transcriptase-polymerase chain reaction for prostate-specific antigen mRNA

Abstract

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 β-actin, a housekeeping gene. The ratio of the amplification products obtained reflects the relative amount of PSA mRNA with respect to actin mRNA. During PCR, digoxigenin-dUTP is incorporated in the amplified sequences. The PCR products are analyzed separately by time-resolved immunofluorometric hybridization assays, using specific probes immobilized in microtiter wells. The hybrids are reacted with alkaline phosphatase-labeled antidigoxigenin antibody. The phosphate ester of fluorosalicylate is used as a substrate. The fluorosalicylate produced forms a fluorescent complex with Tb 3+-EDTA which is measured by time-resolved fluorometry. Results: The hybridization assays for both PSA and actin amplification products show linearity in the range of 1.4–110 pmol/L. The exponential phase of PCR amplification extends up to 200,000 and 100,000 PSA and actin cDNA molecules, respectively. We prepared mixtures 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 when samples containing 150, 300 and 1600 LNCaP cells were analyzed ( n = 4). Conclusions: A quantitative analytical methodology is provided for monitoring PSA mRNA. The assay is expected to be beneficial in the study of prostate cancer spread.