Quantitative Detection of t(14;18)-Positive Cells by Real-Time Quantitative PCR Using Fluorogenic Probes
Open Access
- 1 December 1998
- journal article
- Published by Taylor & Francis in BioTechniques
- Vol. 25 (6) , 1058-1064
- https://doi.org/10.2144/98256cr05
Abstract
To detect t(14;18)-positive cells present in human lymphoma tissue, bone marrow aspirates and peripheral blood mononuclear cells (PBMNC), we have established an automated, real-time quantitative PCR using double-labeled fluorogenic probes. In relation to t(14;18)-positive genomic DNA or a cloned t(14;18)-DNA fragment, highly reproducible results can be obtained with initial copy numbers between 10 to 105. The detection of single copies has been verified by the stochastic multiple-tube approach. PBMNC cells obtained during clinical follow-up of patients with follicular lymphoma were analyzed by the one-step, real-time quantitative PCR and a two-step, semi-nested PCR combined with a limiting dilution assay. The quantitative results obtained by both assays correlate very well. Real-time quantitative PCR has several advantages: (i) it involves less critical pipetting steps, (ii) is less timeconsuming and (iii) UTP, in combination with uracil-N-glycosylase, can be used to control carryover contamination. The h...Keywords
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