DETECTION OF PROSTATE-SPECIFIC ANTIGEN MESSENGER-RNA BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION AND TIME-RESOLVED FLUOROMETRY

We have developed a time-resolved fluorometric hybridization assay for detecting prostate-specific antigen (PSA) mRNA amplified by reverse t ranscription polymerase chain reaction. During PCR, digoxigenin-11-dUT P is incorporated into the amplified product. An oligonucleotide inter nal to the primers is used as a specific probe, being biotinylated and captured on streptavidin-coated microtiter wells. Denatured PCR produ ct hybridizes with the probe, and the hybrids are detected with an alk aline phosphatase-labeled antidigoxigenin antibody, We used the phosph ate ester of fluorosalicylic acid as the substrate. The fluorosalicyla te produced forms a highly fluorescent ternary complex with Tb3(+)-EDT A, which we can measure by time-resolved fluorometry. A signal-to-back ground ratio of 10 was obtained when 160 PSA cDNA molecules were prese nt in the preamplification sample. Also, mRNA corresponding to one LNC aP cell in the presence of 10(6) PSA-negative cells can be detected (s ignal-to-background ratio of 3.1). Samples containing 100, 1000, and 5 0 000 LNCaP cells gave CVs of 12.4%, 4.9%, and 6.8%, respectively (n = 10).