Advanced

Dual-label detection of amplified products in quantitative RT-PCR assay using lanthanide-labeled probes

Ylikoski, Alice; Karp, Matti; Lilja, Hans LU and Lovgren, T (2001) In BioTechniques 30(4). p.832-845
Abstract
Quantitative RT-PCR (QRT-PCR) enables the sensitive and specific detection of mRNA with a small copy number. We used the QRT-PCR method and dual-label analysis of amplification products for the detection of prostate-specific antigen (PSA) mRNA. The QRT-PCR assay employed a PSA-like internal standard (IS) mRNA, which was used to quantify the PSA mRNA copies and to control the variations during the whole assay procedure from the RNA extraction to the detection of QRT-PCR amplification products by hybridization assay. After co-amplification, the PSA and IS products were detected in a microplate using Eu3+ chelate-labeled PSA and Tb3+ chelate-labeled IS hybridization probes. The detection probes allowed the simultaneous and dual-label... (More)
Quantitative RT-PCR (QRT-PCR) enables the sensitive and specific detection of mRNA with a small copy number. We used the QRT-PCR method and dual-label analysis of amplification products for the detection of prostate-specific antigen (PSA) mRNA. The QRT-PCR assay employed a PSA-like internal standard (IS) mRNA, which was used to quantify the PSA mRNA copies and to control the variations during the whole assay procedure from the RNA extraction to the detection of QRT-PCR amplification products by hybridization assay. After co-amplification, the PSA and IS products were detected in a microplate using Eu3+ chelate-labeled PSA and Tb3+ chelate-labeled IS hybridization probes. The detection probes allowed the simultaneous and dual-label detection of PSA and IS products in the same microtiter well. Compared to the single-label assay, the dual-label detection improved the within- and between-assay CV% from 21.7 to 7.5 and from 36.0 to 30.3, respectively. The between- and within-assay variation of the dual-label assay was further studied using PSA-producing LNCaP cells. The cells were found to express 980 +/- 170 (mean +/- SD) copies of PSA-mRNA with the within-assay CV% of 17.7 and 890 +/- 220 (mean +/- SD) copies of PSA-mRNA with the between-assay CV% of 25.0. The methodology developed may help in future studies to obtain reliable quantification of PSA mRNA generated by circulating prostate cancer cells. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
BioTechniques
volume
30
issue
4
pages
832 - 845
publisher
Informa Healthcare
external identifiers
  • pmid:11314266
  • scopus:0035050869
ISSN
0736-6205
language
English
LU publication?
yes
id
e1079308-da2c-4494-97d0-4a79c1f760eb (old id 1121908)
date added to LUP
2008-07-18 16:02:51
date last changed
2018-06-10 04:37:13
@article{e1079308-da2c-4494-97d0-4a79c1f760eb,
  abstract     = {Quantitative RT-PCR (QRT-PCR) enables the sensitive and specific detection of mRNA with a small copy number. We used the QRT-PCR method and dual-label analysis of amplification products for the detection of prostate-specific antigen (PSA) mRNA. The QRT-PCR assay employed a PSA-like internal standard (IS) mRNA, which was used to quantify the PSA mRNA copies and to control the variations during the whole assay procedure from the RNA extraction to the detection of QRT-PCR amplification products by hybridization assay. After co-amplification, the PSA and IS products were detected in a microplate using Eu3+ chelate-labeled PSA and Tb3+ chelate-labeled IS hybridization probes. The detection probes allowed the simultaneous and dual-label detection of PSA and IS products in the same microtiter well. Compared to the single-label assay, the dual-label detection improved the within- and between-assay CV% from 21.7 to 7.5 and from 36.0 to 30.3, respectively. The between- and within-assay variation of the dual-label assay was further studied using PSA-producing LNCaP cells. The cells were found to express 980 +/- 170 (mean +/- SD) copies of PSA-mRNA with the within-assay CV% of 17.7 and 890 +/- 220 (mean +/- SD) copies of PSA-mRNA with the between-assay CV% of 25.0. The methodology developed may help in future studies to obtain reliable quantification of PSA mRNA generated by circulating prostate cancer cells.},
  author       = {Ylikoski, Alice and Karp, Matti and Lilja, Hans and Lovgren, T},
  issn         = {0736-6205},
  language     = {eng},
  number       = {4},
  pages        = {832--845},
  publisher    = {Informa Healthcare},
  series       = {BioTechniques},
  title        = {Dual-label detection of amplified products in quantitative RT-PCR assay using lanthanide-labeled probes},
  volume       = {30},
  year         = {2001},
}