Dual-Label Detection of Amplified Products in Quantitative RT-PCR Assay Using Lanthanide-Labeled Probes

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.

Simultaneous Quantification of Human Glandular Kallikrein 2 and Prostate-Specific Antigen mRNAs in Peripheral Blood from Prostate Cancer Patients

“…9,24 The detection probes for hybridization assay contained additional diaminohexanedeoxycytidines to be labeled with lanthanide chelate as described earlier. [32][33][34] The detection probes for hK2 and PSA were labeled with the Eu 3ϩ chelate, and the probes for the IS-hK2 and IS-PSA were labeled with the Tb 3ϩ chelate.…”

“…The RNA extraction was performed as described earlier. 24 A constant amount of IS-hK2 and IS-PSA mRNA (5 ϫ 10 4 molecules of each) were added into each sample after denaturation of the pelleted cells. Samples containing only 2.5 ϫ 10 6 SP2/0 cells were put up to serve as negative controls in RNA extraction.…”

“…The hybridization was performed as reported previously. 24 Briefly, a 10-l aliquot of PCR product and 50 l of buffer containing 1 mol/L NaCl were added into each streptavidin-coated microtitration well (InnoTrac Diagnostics Oy, Turku, Finland). Each PCR product was added into two wells and the target (PSA or hK2) and IS (IS-PSA or IS-hK2) products were detected from the same wells with the Eu 3ϩ -labeled target and Tb 3ϩ -labeled IS probes, respectively.…”

“…[15][16][17][18][19][20][21] Many of the research groups have stated that the large variations in the results may be due to the various RT-PCR assays each designed and validated differently in the absence of the basis of international standardization of the procedures, thus proposing the need for more standardized and quantitative RT-PCR (QRT-PCR) assays. [22][23][24] The first QRT-PCR assays exploited endogenous RNA standards expressed in the cell. 25 Usually the endoge-nous standard is mRNA expressed by a housekeeping gene, such as ␤-actin or glyceraldehyde 3-phosphate dehydrogenase.…”

“…In addition, if the exogenous mRNA standard is used, it will be possible to control the variations of an assay starting from the RNA extraction step. 9 We have recently presented a QRT-PCR assay for the detection of PSA mRNA 9,24 and our present aim was to develop a QRT-PCR assay for the simultaneous detection of hK2 and PSA mRNAs in blood samples. The QRT-PCR assay uses an external calibration curve and two highly target-like exogenous, internal standard (IS) mRNAs, IShK2 and IS-PSA for the specific quantification of hK2 and PSA mRNAs, respectively.…”