Duplex RT-PCR and chemiluminometric hybridization assay for combined screening of the mRNAs of prostate-specific antigen and prostate-specific membrane antigen in peripheral blood

Abstract

Combined screening of the mRNAs for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) has been proposed as a more useful test than the separate PSA mRNA or PSMA mRNA assays in the molecular diagnosis and monitoring of prostate cancer. We have developed a simple and highly sensitive method for the simultaneous detection of PSA mRNA and PSMA mRNA using duplex RT-PCR and a chemiluminometric hybridization assay. Total RNA from peripheral blood was reverse-transcribed using oligo(dT) 20 primer followed by duplex PCR in the presence of two pairs of primers specific for PSA and PSMA. Heat-denatured biotinylated PCR products were hybridized with PSA- and PSMA-specific oligonucleotide probes immobilized in microtiter wells. The hybrids were determined by using a streptavidin–alkaline phosphatase conjugate and a chemiluminogenic substrate. Using the duplex PCR, 50 copies of PSA DNA and 5 copies of PSMA DNA can be detected with a signal/background ratio of 9.7 and 22, respectively. Analysis of samples containing total RNA corresponding to 0.04–400 LNCaP cells indicated that the detectability of the proposed method is 1 cancer cell equivalent in 10 ml of peripheral blood. The overall reproducibility of the duplex assay, including reverse transcription, PCR, and hybridization assay, ranged from 3.6 to 15.5%. The method is very simple, rapid, sensitive and suitable for high-throughput screening analysis as opposed to the commonly used nested RT-PCR assays with electrophoretic detection.