Novel homogenous time-resolved fluorometric RT-PCR assays for quantification of PSA and hK2 mRNAs in blood.
OBJECTIVE
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.


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.
Rissanen
Maria
M
Department of Biotechnology, University of Turku, Tykistökatu 6A, FIN-20520 Turku, Finland. maria.rissanen@utu.fi
Helo
Pauliina
P
Väänänen
Riina-Minna
RM
Wahlroos
Veikko
V
Lilja
Hans
H
Nurmi
Martti
M
Pettersson
Kim
K
Nurmi
Jussi
J
D005189:False Positive Reactions; D005260:Female; D005470:Fluorometry / Q000379:methods*; D018628:Gene Dosage; D006801:Humans; D008297:Male; D017430:Prostate-Specific Antigen / Q000097:blood* / Q000235:genetics*; D012333:RNA, Messenger / Q000097:blood* / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors; D020840:Tissue Kallikreins / Q000097:blood* / Q000235:genetics*; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D020840:Tissue Kallikreins; D017430:Prostate-Specific Antigen