Cookies on this website

We use cookies to ensure that we give you the best experience on our website. If you click 'Accept all cookies' we'll assume that you are happy to receive all cookies and you won't see this message again. If you click 'Reject all non-essential cookies' only necessary cookies providing core functionality such as security, network management, and accessibility will be enabled. Click 'Find out more' for information on how to change your cookie settings.

OBJECTIVES: The purpose of this study was to design, validate, and optimize internally standardized real-time quantitative RT-PCR assays and to identify and avoid problems with assay reliability and examine the impact of an exogenous internal standard. DESIGN AND METHODS: The model system consisted of internally standardized quantitative real-time RT-PCR assays specific for PSA and hK2 mRNA based on time-resolved fluorometric detection of lanthanide chelates. RESULTS: Reproducibility was best when large copy numbers (>5000 per milliliter blood) were analyzed. Addition of an exogenous target-mimicking internal standard had no significant effect on the reproducibility of the method, but increased the calculated copy numbers by an average of 2-fold. CONCLUSIONS: We developed an internally standardized, specific and reproducible real-time RT-PCR analysis method for PSA and hK2 mRNA in circulating cells in the bloodstream. Both PSA and hK2 assays are sufficiently sensitive to detect two LNCaP cells per milliliter whole blood.

Original publication




Journal article


Clin Biochem

Publication Date





111 - 118


False Positive Reactions, Female, Fluorometry, Gene Dosage, Humans, Male, Prostate-Specific Antigen, RNA, Messenger, Reference Standards, Reproducibility of Results, Reverse Transcriptase Polymerase Chain Reaction, Time Factors, Tissue Kallikreins, Tumor Cells, Cultured