HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Submit an electronic Letter to the Editor about this paper Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (27) Citing Articles via Google Scholar Google Scholar Articles by Ylikoski, A. Articles by Iitiä, A. Search for Related Content PubMed PubMed Citation Articles by Ylikoski, A. Articles by Iitiä, A. Related Collections Molecular Diagnostics and Genetics

Quantitative Reverse Transcription-PCR Assay with an Internal Standard for the Detection of Prostate-specific Antigen mRNA

¹ Department of Biotechnology, University of Turku, Tykistökatu 6 A, 6th Floor, FIN-20520 Turku, Finland.

² Department of Clinical Chemistry, Lund University, University Hospital, S-20502 Malmö, Sweden.

³ InnoTrac Diagnostics Oy, Tykistökatu 6 A, 7th Floor, FIN-20520 Turku, Finland.
^a Author for correspondence. Fax 358-2-3338050; e-mail alice.ylikoski{at}utu.fi

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 10⁶ 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 ~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.

The following articles in journals at HighWire Press have cited this article:

				D. R. Shaffer, M. A. Leversha, D. C. Danila, O. Lin, R. Gonzalez-Espinoza, B. Gu, A. Anand, K. Smith, P. Maslak, G. V. Doyle, et al. Circulating Tumor Cell Analysis in Patients with Progressive Castration-Resistant Prostate Cancer Clin. Cancer Res., April 1, 2007; 13(7): 2023 - 2029. [Abstract] [Full Text] [PDF]

				M. P.M.Q. van Gils, D. Hessels, O. van Hooij, S. A. Jannink, W. P. Peelen, S. L.J. Hanssen, J. A. Witjes, E. B. Cornel, H. F.M. Karthaus, G. A.H.J. Smits, et al. The Time-Resolved Fluorescence-Based PCA3 Test on Urinary Sediments after Digital Rectal Examination; a Dutch Multicenter Validation of the Diagnostic Performance Clin. Cancer Res., February 1, 2007; 13(3): 939 - 943. [Abstract] [Full Text] [PDF]

				J. Groskopf, S. M.J. Aubin, I. L. Deras, A. Blase, S. Bodrug, C. Clark, S. Brentano, J. Mathis, J. Pham, T. Meyer, et al. APTIMA PCA3 Molecular Urine Test: Development of a Method to Aid in the Diagnosis of Prostate Cancer Clin. Chem., June 1, 2006; 52(6): 1089 - 1095. [Abstract] [Full Text] [PDF]

				D. Schamhart, J. Swinnen, K.-H. Kurth, A. Westerhof, R. Kusters, H. Borchers, and C. Sternberg Numeric Definition of the Clinical Performance of the Nested Reverse Transcription-PCR for Detection of Hematogenous Epithelial Cells and Correction for Specific mRNA of Non-Target Cell Origin as Evaluated for Prostate Cancer Cells Clin. Chem., September 1, 2003; 49(9): 1458 - 1466. [Abstract] [Full Text] [PDF]

				A. Ylikoski, K. Pettersson, J. Nurmi, K. Irjala, M. Karp, H. Lilja, T. Lovgren, and M. Nurmi Simultaneous Quantification of Prostate-specific Antigen and Human Glandular Kallikrein 2 mRNA in Blood Samples from Patients with Prostate Cancer and Benign Disease Clin. Chem., August 1, 2002; 48(8): 1265 - 1271. [Abstract] [Full Text] [PDF]

				F. Savagner, P. Rodien, P. Reynier, V. Rohmer, J.-C. Bigorgne, and Y. Malthiery Analysis of Tg Transcripts by Real-Time RT-PCR in the Blood of Thyroid Cancer Patients J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 635 - 639. [Abstract] [Full Text] [PDF]

				A. Ylikoski, M. Karp, K. Pettersson, H. Lilja, and T. Lovgren Simultaneous Quantification of Human Glandular Kallikrein 2 and Prostate-Specific Antigen mRNAs in Peripheral Blood from Prostate Cancer Patients J. Mol. Diagn., August 1, 2001; 3(3): 111 - 122. [Abstract] [Full Text] [PDF]

				B. S. Sorensen, H. Schmidt, H. von der Maase, P. T. Straten, and E. Nexo Quantification of Melanoma Cell-specific MART-1 mRNA in Peripheral Blood by a Calibrated Competitive Reverse Transcription-PCR Clin. Chem., December 1, 2000; 46(12): 1923 - 1928. [Abstract] [Full Text] [PDF]

				J. Nurmi, A. Ylikoski, T. Soukka, M. Karp, and T. Lovgren A new label technology for the detection of specific polymerase chain reaction products in a closed tube Nucleic Acids Res., April 15, 2000; 28(8): e28 - e. [Abstract] [Full Text] [PDF]