Simultaneous detection of two cystic fibrosis alleles using dual-label time-resolved fluorometry

Litiä, Antti; Liukkonen, Liisa; Siitari, Harri

doi:10.1016/0890-8508(92)90047-2

Cited by 18 publications

(6 citation statements)

References 30 publications

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“…The use of different lanthanide chelates permits the detection of multiple analytes in the same reaction because of the sharp emission peaks and the different decay times of the lanthanides (Figure 1). The TRF technology and the detection of multiple analytes have been widely used in both hybridization assays for DNA and immunoassays (13,14,20,29,31,38,43). One of the most multiplexed assays has employed seven colors in TRF-based hybridization analysis (30).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2001

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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.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2001

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“…The oligonucleotides used in the hybridization assay ( Table 1) were labelled with Eu‐chelate and biotinylated according to Iitiä et al . (1992 ).…”

Section: Methodsmentioning

confidence: 99%

“…The oligonucleotides used in the hybridization assay (Table 1) were labelled with Eu-chelate and biotinylated according to Iitiä et al (1992). A diaminohexane modified deoxycytidine phosphoramidite (Sund et al, 1988) was used to introduce amino functions to the oligonucleotides.…”

Section: Hybridization Assaymentioning

confidence: 99%

Determination of a common genetic variant of luteinizing hormone using DNA hybridization and immunoassays

Nilsson

Jiang

Pettersson

et al. 1998

Clinical Endocrinology

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“…Generally, in TRF-based hybridisation assays the sensitivities of short probes have varied from 2 x 10 7 to 1 x 10 10 molecules [10,15,30], depending on the lanthanide label and the length of the probe used. The assay is unable to distinguish DQA1*0101 from *0104, *0301 from *0302 and *0501 from *0503 allele.…”

Section: Discussionmentioning

confidence: 99%

“…The label is detected after a time delay, when background fluorescence has disappeared, which increases the sensitivity of the system. TRF has previously been successfully applied to many different microtitration plate based solution hybridisation assays [6,9,10,11,[13][14][15]16].…”

Section: Introductionmentioning

confidence: 99%

Time‐Resolved Fluorometry Based Sandwich Hybridisation Assay for HLA‐DQA1 Typing

et al. 1998

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ABSTRACT:A microtitration plate based timeresolved fluorescence (TRF) hybridisation assay was developed for HLA typing utilising biotinylated sequence-specific catching probes and europium (Eu) labelled gene locus-specific detection probe to allow time-resolved fluorometer reading of the reaction. In an application for HLA-DQA typing a 228 base pair long region of the polymorphic exon 2 of DQA1 gene was amplified and the denatured PCR product distributed into streptavidin-coated microtitration wells together with the detection probe and one of the catching probes. After incubation and washes, the enhancement solution was added and specific hybridisation signal detected by measuring the emitted light. A series of 100 isolated genomic DNA samples were studied using biotinylated probes specific for DQA1*01, *0101/0104, *0103/0201/0601, *0201, *03, *0401/0601, *05 and *0502 alleles with results demonstrating the capacity of the test to detect aimed alleles. A series of whole blood spot samples were also studied and the results confirmed the applicability of this modification of the test.

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Simultaneous detection of two cystic fibrosis alleles using dual-label time-resolved fluorometry

Cited by 18 publications

References 30 publications

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

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

Determination of a common genetic variant of luteinizing hormone using DNA hybridization and immunoassays

Time‐Resolved Fluorometry Based Sandwich Hybridisation Assay for HLA‐DQA1 Typing

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