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

Ylikoski, A.; Sjöroos, M.; Lundwall, Åke LU ; Karp, M.; Lövgren, T.; Lilja, Hans LU and Iitia, A. (1999) In Clin Chem 45(9). p.1397-407
Abstract
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... (More)
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. (Less)
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author
publishing date
type
Contribution to journal
publication status
published
subject
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
in
Clin Chem
volume
45
issue
9
pages
1397 - 407
external identifiers
  • Scopus:0032871448
language
English
LU publication?
no
id
e9033867-4c9a-4c1d-9087-de8a1c37fcbf (old id 3966325)
alternative location
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10471641
date added to LUP
2013-08-11 14:34:33
date last changed
2016-06-29 09:03:18
@misc{e9033867-4c9a-4c1d-9087-de8a1c37fcbf,
  abstract     = {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.},
  author       = {Ylikoski, A. and Sjöroos, M. and Lundwall, Åke and Karp, M. and Lövgren, T. and Lilja, Hans and Iitia, A.},
  keyword      = {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},
  language     = {eng},
  number       = {9},
  pages        = {1397--407},
  series       = {Clin Chem},
  title        = {Quantitative reverse transcription-PCR assay with an internal standard for the detection of prostate-specific antigen mRNA},
  volume       = {45},
  year         = {1999},
}