QUANTIFICATION OF PROSTATE-SPECIFIC ANTIGEN MESSENGER-RNA BY COAMPLIFICATION WITH A RECOMBINANT RNA INTERNAL STANDARD AND MICROTITER WELL-BASED HYBRIDIZATION

We report a quantitative analytical methodology for prostate-specific antigen (PSA) mRNA, which is based on the coamplification of the targe t with a recombinant RNA internal standard (IS) using reverse transcri ptase-polymerase chain reaction. PSA mRNA and the RNA IS contain the s ame primer recognition sites and generate amplification products that have identical sizes but differ in a 24-bp sequence located in the cen ter of the molecule. Amplified sequences are labeled with biotin using a biotinylated upstream primer. The products are captured on streptav idin-coated microtiter wells and hybridized to specific probes labeled with the hapten digoxigenin. The hybrids are determined using alkalin e phosphatase-labeled anti-digoxigenin antibody and time-resolved fluo rometry. The ratio of the fluorescence values obtained for the PSA mRN A and the RNA IS is a linear function of the amount of PSA mRNA presen t in the sample. Samples containing total RNA from PSA-expressing cell s (LNCaP cells) in addition to 1 mu g of RNA from healthy cells give f luorescence ratios related linearly to the number of cells in the rang e of 4 to 3000 cells.