Novel homogenous time-resolved fluorometric RT-PCR assays for quantification of PSA and hK2 mRNAs in blood Journal Article
| Authors: | Rissanen, M.; Helo, P.; Väänänen, R. M.; Wahlroos, V.; Lilja, H.; Nurmi, M.; Pettersson, K.; Nurmi, J. |
| Article Title: | Novel homogenous time-resolved fluorometric RT-PCR assays for quantification of PSA and hK2 mRNAs in blood |
| Abstract: | 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. © 2006. |
| Keywords: | controlled study; human cell; sensitivity and specificity; reproducibility; prostate specific antigen; reproducibility of results; reverse transcription polymerase chain reaction; tumor cells, cultured; validation study; time factors; prostate-specific antigen; standard; messenger rna; reference standards; reverse transcriptase polymerase chain reaction; rna, messenger; quantitative analysis; antigen detection; blood analysis; reliability; gene dosage; false positive reactions; chelate; cell strain lncap; calculation; fluorometry; tissue kallikreins; process development; blood chemistry; process design; neoplasm circulating cells; lanthanide |
| Journal Title: | Clinical Biochemistry |
| Volume: | 40 |
| Issue: | 1-2 |
| ISSN: | 0009-9120 |
| Publisher: | Elsevier Inc. |
| Date Published: | 2007-01-01 |
| Start Page: | 111 |
| End Page: | 118 |
| Language: | English |
| DOI: | 10.1016/j.clinbiochem.2006.10.005 |
| PUBMED: | 17126312 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 5" - "Export Date: 17 November 2011" - "CODEN: CLBIA" - "Source: Scopus" |
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