Quantification of prostate-specific antigen mRNA by coamplification with a recombinant RNA internal standard and microtiter well-based hybridization.
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 microg of RNA from healthy cells give fluorescence ratios related linearly to the number of cells in the range of 4 to 3000 cells.
Verhaegen
M
M
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Ioannou
P C
PC
Christopoulos
T K
TK
D000230:Adenocarcinoma / Q000201:enzymology* / Q000473:pathology; D005470:Fluorometry; D006801:Humans; D008297:Male; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D017430:Prostate-Specific Antigen / Q000235:genetics / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000201:enzymology* / Q000473:pathology; D012333:RNA, Messenger / Q000096:biosynthesis*; D012334:RNA, Neoplasm / Q000096:biosynthesis / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011994:Recombinant Proteins; D017430:Prostate-Specific Antigen