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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

DOI

10.1016/j.clinbiochem.2006.10.005

Type

Journal article

Journal

Clin Biochem

Publication Date

01/2007

Volume

40

Pages

111 - 118

Keywords

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