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High-throughput chemiluminometric determination of prostate-specific membrane antigen mRNA in peripheral blood by RT-PCR using a synthetic RNA internal standard

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Abstract

A quantitative reverse transcriptase polymerase chain reaction (RT-PCR) method, employing internal standard (IS) RNA and a simplified chemiluminometric hybridization assay, is described for the determination of prostate-specific membrane antigen (PSMA) mRNA. The recombinant RNA IS has the same binding sites and size as the amplified PSMA mRNA. Biotinylated PCR products (263 bp) from PSMA mRNA and RNA IS are captured in microtiter wells coated with streptavidin, and hybridized with alkaline phosphatase-conjugated probes. The bound alkaline phosphatase (AP) is measured by using a chemiluminogenic substrate. The ratio of the luminescence values obtained for PSMA mRNA and the RNA IS is a linear function of the initial amount of PSMA mRNA present in the sample before RT-PCR. The linear range extends from 500 to 5,000,000 PSMA mRNA copies and the overall reproducibility of the assay, including RT-PCR and hybridization, ranges from 7.4 to 16.6%. Samples containing total RNA from PSMA-expressing LNCaP cells give luminescence ratios linearly related to the number of cells in the range 0.5–5,000 cells.

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Acknowledgements

Financial support by a research grant (YPER) from the General Secretariat for Research and Technology (Greece) and Medicon Hellas SA is gratefully acknowledged.

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Authors and Affiliations

  1. Department of Chemistry, University of Athens, 15771, Athens, Greece

    Evaggelia Emmanouilidou & Penelope C. Ioannou

  2. Department of Chemistry, University of Patras, 26500, Patras, Greece

    Theodore K. Christopoulos

  3. Institute of Chemical Engineering and High Temperature Chemical Processes, P.O. Box 1414, 26500, Patras, Greece

    Theodore K. Christopoulos

Authors
  1. Evaggelia Emmanouilidou
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  2. Penelope C. Ioannou
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  3. Theodore K. Christopoulos
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Correspondence to Penelope C. Ioannou.

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Emmanouilidou, E., Ioannou, P.C. & Christopoulos, T.K. High-throughput chemiluminometric determination of prostate-specific membrane antigen mRNA in peripheral blood by RT-PCR using a synthetic RNA internal standard. Anal Bioanal Chem 380, 90–97 (2004). https://doi.org/10.1007/s00216-004-2719-6

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  • DOI: https://doi.org/10.1007/s00216-004-2719-6

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