Improved reverse transcriptase-polymerase chain reaction protocol with exogenous internal competitive control for prostate-specific antigen mRNA in blood and bone marrow.
The possibility of improving diagnosis of micrometastases from prostate cancer by further enhancing the detection of prostate-specific antigen-producing cells in circulation is being evaluated. We have developed a reverse transcriptase-PCR protocol with the desirable characteristics of low limit of detection, high specificity, reproducibility of response, and ease of performance. Among the procedural alterations that have contributed to these improvements are longer PCR primers, a two-step amplification cycle, and hot-start PCR. We have lowered the limit of detection to one LNCaP prostate-cancer cell in 10(8) peripheral blood mononuclear cells, and samples of blood and bone marrow from healthy donors have yielded no false positives. Because PCR procedures frequently exhibit tube-to-tube variability, we have incorporated a set of internal and external controls into the protocol-a significant advance in assuring assay reliability.
Corey
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Department of Urology, School of Medicine, University of Washington, Seattle 98195, USA. ecorey@u.washington.edu
Arfman
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Liu
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Vessella
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D001853:Bone Marrow / Q000473:pathology*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000473:pathology; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D017430:Prostate-Specific Antigen / Q000097:blood* / Q000235:genetics; D011471:Prostatic Neoplasms / Q000097:blood* / Q000175:diagnosis; D012333:RNA, Messenger / Q000097:blood*; D012334:RNA, Neoplasm / Q000097:blood; D012194:RNA-Directed DNA Polymerase; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012194:RNA-Directed DNA Polymerase; D017430:Prostate-Specific Antigen