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Quantitative reverse transcription-PCR assay with an internal standard for the detection of prostate-specific antigen mRNA

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Author

Summary, in English

BACKGROUND: Circulating prostate cells can be detected with a reverse transcription-PCR (RT-PCR) assay for prostate-specific antigen (PSA) mRNA. We have developed a new quantitative RT-PCR method for measuring PSA mRNA. METHODS: The method uses a PSA-like internal standard (IS) mRNA that is added into the sample at the beginning of the RNA extraction and coamplified by RT-PCR with the PSA in the sample. After PCR amplification, the IS and PSA products are selectively detected by hybridization in a microtitration plate using probes labeled with fluorescent europium chelates. RESULTS: The method was validated with PSA and IS mRNAs and PSA-expressing cells to obtain a detection limit of 50 PSA mRNA copies (i.e., signal 2 times the mean of zero signal), linearity up to 10(6) copies, and detection of a single PSA-expressing cell. In preliminary evaluations, 60% (n = 10) of the prostate cancer patients with skeletal metastases gave results above the detection limit (500 PSA mRNA copies in 5 mL of blood). The total number of PSA copies ranged from 900 +/- 200 to 44 100 +/- 4900 (mean +/- SD) in the samples, corresponding to approximately 1-100 PSA-expressing cells in 5 mL of blood. In the controls (n = 34), none of the healthy females and 2 of 19 healthy males had detectable PSA mRNA [700 +/- 100 and 2000 +/- 900 (mean +/- SD) PSA mRNA copies in 5 mL of blood for the 2 males]. CONCLUSIONS: The assay provides sensitive and quantitative detection of PSA mRNA expression from blood samples and can be used to establish the clinically significant number of PSA mRNA copies in prostate cancer.

Publishing year

1999

Language

English

Pages

407-1397

Publication/Series

Clinical Chemistry

Volume

45

Issue

9

Document type

Journal article

Publisher

American Association for Clinical Chemistry

Topic

  • Medicinal Chemistry

Keywords

  • Sensitivity and Specificity
  • Reverse Transcriptase Polymerase Chain Reaction/methods
  • Non-U.S. Gov't
  • Research Support
  • Reproducibility of Results
  • Messenger/*analysis/blood/metabolism
  • RNA
  • Prostatic Neoplasms/blood
  • Prostate-Specific Antigen/*analysis/blood/metabolism
  • Male
  • Humans
  • Cell Line
  • Tumor Markers
  • Biological/*analysis/blood/metabolism

Status

Published

ISBN/ISSN/Other

  • ISSN: 0009-9147