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

Rissanen, Maria; Helo, Pauliina; Vaananen, Riina-Minna; Wahlroos, Veikko; Lilja, Hans LU ; Nurmi, Martti; Pettersson, Kim and Nurmi, Jussi (2007) In Clinical Biochemistry 40(1-2). p.111-118
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... (More)
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. (c) 2006 The Canadian Society of Clinical Chemists. All rights reserved. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
sensitivity and, tissue kallikreins, specificity, reverse transcriptase polymerase chain reaction, neoplasm circulating cells, reproducibility of results
in
Clinical Biochemistry
volume
40
issue
1-2
pages
111 - 118
publisher
Elsevier
external identifiers
  • wos:000243631100019
  • scopus:33845764381
ISSN
1873-2933
DOI
10.1016/j.clinbiochem.2006.10.005
language
English
LU publication?
yes
id
c5a54b8c-99ea-47dc-81ed-85464ef1ccd4 (old id 676706)
date added to LUP
2007-12-20 14:44:49
date last changed
2017-01-01 04:20:12
@article{c5a54b8c-99ea-47dc-81ed-85464ef1ccd4,
  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. (c) 2006 The Canadian Society of Clinical Chemists. All rights reserved.},
  author       = {Rissanen, Maria and Helo, Pauliina and Vaananen, Riina-Minna and Wahlroos, Veikko and Lilja, Hans and Nurmi, Martti and Pettersson, Kim and Nurmi, Jussi},
  issn         = {1873-2933},
  keyword      = {sensitivity and,tissue kallikreins,specificity,reverse transcriptase polymerase chain reaction,neoplasm circulating cells,reproducibility of results},
  language     = {eng},
  number       = {1-2},
  pages        = {111--118},
  publisher    = {Elsevier},
  series       = {Clinical Biochemistry},
  title        = {Novel homogenous time-resolved fluorometric RT-PCR assays for quantification of PSA and hK2 mRNAs in blood},
  url          = {http://dx.doi.org/10.1016/j.clinbiochem.2006.10.005},
  volume       = {40},
  year         = {2007},
}