PubMed · 5669300
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A B Kaulin, V K Fokin. 1968. [Polarization microfluorimeter].. https://pubmed.ncbi.nlm.nih.gov/5669300/
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We have developed a time-resolved fluorometric hybridization assay for detecting prostate-specific antigen (PSA) mRNA amplified by reverse transcription polymerase chain reaction. During PCR, digoxigenin-11-dUTP is incorporated into the amplified product. An oligonucleotide internal to the primers is used as a specific probe, being biotinylated and captured on streptavidin-coated microtiter wells. Denatured PCR product hybridizes with the probe, and the hybrids are detected with an alkaline phosphatase-labeled antidigoxigenin antibody. We used the phosphate ester of fluorosalicylic acid as the substrate. The fluorosalicylate produced forms a highly fluorescent ternary complex with Tb(3+)-EDTA, which we can measure by time-resolved fluorometry. A signal-to-background ratio of 10 was obtained when 160 PSA cDNA molecules were present in the preamplification sample. Also, mRNA corresponding to one LNCaP cell in the presence of 10(6) PSA-negative cells can be detected (signal-to-background ratio of 3.1). Samples containing 100, 1000, and 50,000 LNCaP cells gave CVs of 12.4%, 4.9%, and 6.8%, respectively (n = 10).
In vitro lymphocyte mitogenic stimulation by phytohemagglutinin A was determined in 20 subjects, comparing tritiated thymidine incorporation and nuclear propidium iodide fluorescence. Unexpectedly, no correlation could be found between the two measures. The pitfalls of both methods are reviewed and discussed. The inability to validate one test by the other restricts the interpretation of the results obtained with one method and prevents their generalization. Without another gold-standard at the present time, specific and limited method-dependent norms should be defined.
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