scholarly journals Detection of prostate-specific antigen mRNA and protein in breast tumors

1996 ◽  
Vol 42 (3) ◽  
pp. 361-366 ◽  
Author(s):  
N Zarghami ◽  
E P Diamandis
Keyword(s):  
Polymerase Chain Reaction ◽  
Prostate Specific Antigen ◽  
Reverse Transcription ◽  
Specific Antigen ◽  
Breast Tumors ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Highly Sensitive ◽  
Polymerase Chain ◽  
Good Agreement

Abstract We have developed reverse transcription-polymerase chain reaction (RT- PCR) methods for detecting prostate-specific antigen (PSA) mRNA. Using these methods, and a highly sensitive immunofluorometric assay for measuring PSA protein, we have assessed the concentrations of PSA mRNA and PSA protein in 30 primary breast tumors and a few other control tissues. We found good agreement between presence of PSA protein and PSA mRNA in breast tumors. We thus propose that, in women, detection of PSA protein or PSA mRNA in tissues and tumors offers equivalent information. Because PSA protein is present in male blood and thus could contaminate extracts from tumors and tissues from men, we propose that the RT-PCR methods we describe be used to assess nonprostatic expression of the PSA gene in men.

Detection of prostate-specific antigen mRNA by reverse transcription polymerase chain reaction and time-resolved fluorometry

1995 ◽  
Vol 41 (12) ◽  
pp. 1705-1709 ◽  
Author(s):  
B Galvan ◽  
T K Christopoulos ◽  
E P Diamandis
Keyword(s):  
Polymerase Chain Reaction ◽  
Prostate Specific Antigen ◽  
Reverse Transcription ◽  
Specific Antigen ◽  
Phosphate Ester ◽  
Chain Reaction ◽  
Time Resolved ◽  
Background Ratio ◽  
Pcr Product ◽  
Polymerase Chain

Abstract 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).

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