No abstract

A novel signal generation principle suitable for real time and end-point detection of specific PCR products in a closed tube is described. Linear DNA probes were labeled at their 5′-ends with a stable, fluorescent terbium chelate. The fluorescence intensity of this chelate is lower when it is coupled to single-stranded DNA than when the chelate is free in solution. The synthesized probes were used in the real time monitoring of PCR using a prototype instrument that consisted of a fluorometer coupled to a thermal cycler. When the probe anneals to a complementary target amplicon, the 5′→3′ exonucleolytic activity of DNA polymerase detaches the label from the probe. This results in an enhanced terbium fluorescence signal. Since terbium has a long excited state lifetime, its fluorescence can be measured in a time-resolved manner, which results in a low background fluorescence and a 1000-fold signal amplification. The detection method is quantitative over an extremely wide linear range (at least 10-10 7 initial template molecules). The label strategy can easily be combined with existing label technologies, such as TaqMan 5′-exonuclease assays, in order to carry out multiplex assays that do not suffer from overlapping emission peaks of the fluorophores.

Two time-resolved fluorescence-based methods for nucleic acid quantification are described and their results are compared. Both methods use an exogenous internal standard to eliminate errors arising from different steps of the assay. The first method is a competitive end-point assay, where the standard competes for the same primers with the actual target sequence, prostate-specific antigen (PSA) cDNA. The standard and target are quantified in a dual-label plate hybridization with lanthanide-labelled probes after a fixed number of PCR cycles. The second method is based on real-time monitoring of PCR and on the use of a novel homogeneous signal generation principle that relies on the use of a 5'-->3' exonucleolytic DNA polymerase and a probe labelled with an environment sensitive, stable and fluorescent lanthanide chelate. In this assay, a non-competitive, exogenous internal standard is used. Both assays have a wide linear range (50-5 x 10(6) and 10-5 x 10(7) input PSA cDNA molecules for the end-point and real-time assays, respectively) and there is a strong correlation between the results obtained with the two assays (r = 1.0). Being somewhat faster to perform, the real-time format is better suited for assays that require high throughput.

No abstract

Background: Circulating prostate cells can be detected with a reverse transcription-PCR (RT-PCR) assay for prostate-specific antigen (PSA) mRNA. We have developed a new quantitative RT-PCR method for measuring PSA mRNA. Methods: The method uses a PSA-like internal standard (IS) mRNA that is added into the sample at the beginning of the RNA extraction and coamplified by RT-PCR with the PSA in the sample. After PCR amplification, the IS and PSA products are selectively detected by hybridization in a microtitration plate using probes labeled with fluorescent europium chelates. Results: The method was validated with PSA and IS mRNAs and PSA-expressing cells to obtain a detection limit of 50 PSA mRNA copies (i.e., signal 2 times the mean of zero signal), linearity up to 106 copies, and detection of a single PSA-expressing cell. In preliminary evaluations, 60% (n = 10) of the prostate cancer patients with skeletal metastases gave results above the detection limit (500 PSA mRNA copies in 5 mL of blood). The total number of PSA copies ranged from 900 ± 200 to 44 100 ± 4900 (mean ± SD) in the samples, corresponding to ∼1–100 PSA-expressing cells in 5 mL of blood. In the controls (n = 34), none of the healthy females and 2 of 19 healthy males had detectable PSA mRNA [700 ± 100 and 2000 ± 900 (mean ± SD) PSA mRNA copies in 5 mL of blood for the 2 males]. Conclusions: The assay provides sensitive and quantitative detection of PSA mRNA expression from blood samples and can be used to establish the clinically significant number of PSA mRNA copies in prostate cancer.

No 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.

Reactive oxygen species are highly reactive molecules and have been implicated in the pathophysiology of many diseases, including diabetes mellitus, cancer, rheumatoid arthritis, and cardiovascular, renal, inflammatory, infectious, and neurologic diseases (1, 2 ). Cells and biological fluids have an array of protective antioxidant mechanisms, both for preventing the production of free radicals and for repairing oxidative damage (3 ). These antioxidant systems include enzymes, macromolecules, and small molecules, including ascorbic acid, ␣-tocopherol, ␤-carotene, ubiquinol-10, reduced lipoic acid (DHLA), reduced glutathione (GSH), methionine, uric acid, bilirubin, and some amino acids. Antioxidants within cells, cell membranes, and extracellular fluids can be up-regulated and mobilized to neutralize excessive and inappropriate formation of reactive oxygen species, but a deficiency of antioxidant defense may lead to a situation of increased oxidative stress.Assays that measure the combined antioxidant effect of the nonenzymatic defenses in biological fluids may be useful in providing an index of ability to resist oxidative damage. Several methods (4 -6 ) have been developed to assess the total antioxidant capacity of human serum or plasma because of the difficulty in measuring each antioxidant component separately and the interactions among different antioxidant components in the serum or plasma. However, the measured antioxidant capacity of a sample depends on which technology and which free radical generator or oxidant is used in the measurement. The ferric reducing ability of plasma (FRAP) assay uses an easily reduced oxidant in a redox-linked colorimetric method (6 ). Because the redox potential of the ferrous/ ferric couple is 0.77 V, any substance or antioxidant with a redox potential Ͻ0.77 V will drive the ferric reduction, assuming stability of redox product. According to a study by Cao and Prior (7 ), the FRAP assay does not measure serum proteins and excludes the low-molecular-weight SH-group-containing antioxidants, such as GSH, DHLA, and some amino acids. We developed a novel method for measuring the ubiquinone-9-reducing ability of plasma (URAP). This new method estimates antioxidant systems with redox potentials Յ0.1 V, which will drive the reduction of ubiquinone-9 (Fig. 1A).HPLC analysis was performed with an ESA Model 582 Solvent Delivery Module, AS1000 autosampler (Thermo Separation Products), and a reversed-phase Microsorb-MV column [150 ϫ 4.6 mm (i.d.); 5-m bead size] from Rainin. The mobile phase consisted of a mixture of anhydrous sodium acetate (4.2 g; Sigma), 15 mL of glacial acetic acid (Mallinckrodt), 15 mL of 2-propanol (Mallinckrodt), 720 mL of methanol (Fisher), and 250 mL of hexane (Fisher). The flow rate was 1.1 mL/min. The electrochemical detector with ESA Model 5200A CouloChem II has been described previously (8 ). The electrochemical cells were a postcolumn guard cell and analytical cell containing dual electrodes in series. The potential of the postcolumn guard cell was set at ϩ0...