High-throughput chemiluminometric determination of prostate-specific membrane antigen mRNA in peripheral blood by RT-PCR using a synthetic RNA internal standard.
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.
Department of Chemistry, University of Athens, 15771 Athens, Greece.
Evaggelia
E
Emmanouilidou
Penelope C
PC
Ioannou
Theodore K
TK
Christopoulos
D000469:Alkaline Phosphatase / Q000737:chemistry; D000954:Antigens, Surface / Q000097:blood* / Q000235:genetics; D002138:Calibration; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry; D005260:Female; D006801:Humans; D008163:Luminescent Measurements / Q000592:standards; D015394:Molecular Structure; D009841:Oligonucleotides / Q000737:chemistry; D012313:RNA / Q000138:chemical synthesis* / Q000737:chemistry / Q000592:standards*; D012333:RNA, Messenger / Q000097:blood* / Q000592:standards*; D011994:Recombinant Proteins / Q000737:chemistry; D012015:Reference Standards; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity
D000954:Antigens, Surface; D009841:Oligonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D000469:Alkaline Phosphatase