Quantitative reverse transcription-PCR assay with an internal standard forthe detection of prostate-specific antigen mRNA

Background: Circulating prostate cells can be detected with a reverse trans cription-PCR (RT-PCR) assay for prostate-specific antigen (PSA) mRNA. We ha ve developed a new quantitative RT-PCR method for measuring PSA mRNA. Methods: The method uses a PSA-like internal standard (IS) mRNA that is add ed into the sample at the beginning of the RNA extraction and coamplified b y RT-PCR with the PSA in the sample. After PCR amplification, the IS and PS A products are selectively detected by hybridization in a microtitration pl ate using probes labeled with fluorescent europium chelates. Results: The method was validated with PSA and IS mRNAs and PSA-expressing cells to obtain a detection limit of 50 PSA mRNA copies (i.e., signal 2 tim es the mean of zero signal), linearity up to 10(6) copies, and detection of a single PSA-expressing cell. In preliminary evaluations, 60% (n = 10) of the prostate cancer patients with skeletal metastases gave results above th e detection limit (500 PSA mRNA copies in 5 nit of blood). The total number of PSA copies ranged from 900 +/- 200 to 44 100 +/- 4900 (mean +/- SD) in the samples, corresponding to similar to 1-100 PSA-expressing cells in 5 mt of blood. In the controls (n = 34), none of the healthy females and 2 of 1 9 healthy males had detectable PSA mRNA [700 +/- 100 and 2000 +/- 900 (mean +/- SD) PSA mRNA copies in 5 mt of blood for the 2 males]. Conclusions: The assay provides sensitive and quantitative detection of PSA mRNA expression from blood samples and can be used to establish the clinic ally significant number of PSA mRNA copies in prostate cancer. (C) 1999 Ame rican Association for Clinical Chemistry.