Dual-label detection of amplified products in quantitative RT-PCR assay using lanthanide-labeled probes

Quantitative RT-PCR (QRT-PCR) enables the sensitive and specific detection of mRNA with a small copy number. We used the QRT-PCR method and dual-label analysis of amplification products for the detection of prostate-specific antigen (PSA) mRNA. The QRT-PCR assay employed a PSA-like internal standard (IS) mRNA, which was used to quantify the PSA mRNA copies and to control t he variations during the whole assay procedure from the RNA extraction to t he detection of QRT-PCR amplification products by hybridization assay. Afte r co-amplification, the PSA and IS products were detected in a microplate u sing Eu3+ chelate-labeled PSA and Tb3+ chelate-labeled IS hybridization pro bes. The detection probes allowed the simultaneous and dual-label detection of PSA and IS products in the same microtiter well. Compared to the single -label assay, the dual-label detection improved the within- and between-ass ay cv% from 21.7 to 7.5 and from 36.0 to 30.3 respectively. The between- an d within-assay variation of the dual-label assay was further studied using PSA-producing LNCaP cells. The cells were found to express 980 +/- 170 (mea n +/- SD) copies of PSA-mRNA with the within-assay cv% of 17.7 and 890 +/- 220 (mean +/- SD) copies of PSA-RNA with the between-assay cv% of 25.0. The methodology developed may help in future studies to obtain reliable quanti fication of PSA mRNA generated by circulating prostate cancer cells.