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Clinical Chemistry 44: 1170-1176, 1998;
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(Clinical Chemistry. 1998;44:1170-1176.)
© 1998 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Quantification of prostate-specific antigen mRNA by coamplification with a recombinant RNA internal standard and microtiterwell-based hybridization

Monique Verhaegen1, Pinelopi C. Ioannou2, and Theodore K. Christopoulos1,a

1 Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada.

2 Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens 15771, Greece.
a Author for correspondence. Fax 519-973-7098; e-mail tkc{at}uwindsor.ca.

We report a quantitative analytical methodology for prostate-specific antigen (PSA) mRNA, which is based on the coamplification of the target with a recombinant RNA internal standard (IS) using reverse transcriptase-polymerase chain reaction. PSA mRNA and the RNA IS contain the same primer recognition sites and generate amplification products that have identical sizes but differ in a 24-bp sequence located in the center of the molecule. Amplified sequences are labeled with biotin using a biotinylated upstream primer. The products are captured on streptavidin-coated microtiter wells and hybridized to specific probes labeled with the hapten digoxigenin. The hybrids are determined using alkaline phosphatase-labeled anti-digoxigenin antibody and time-resolved fluorometry. The ratio of the fluorescence values obtained for the PSA mRNA and the RNA IS is a linear function of the amount of PSA mRNA present in the sample. Samples containing total RNA from PSA-expressing cells (LNCaP cells) in addition to 1 µg of RNA from healthy cells give fluorescence ratios related linearly to the number of cells in the range of 4 to 3000 cells.




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