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• W2137060355 abstract "Abstract Background: Competitive PCR of reverse transcribed mRNA sequences is used to quantify transcripts, but the usual approaches are labor-intensive and time-consuming. We describe the non-gel-based quantification of competitive reverse transcription (RT)-PCR products with use of microparticles and flow cytometry. Methods: PCR products of a target sequence and an internal control sequence (competitor) were labeled during PCR using digoxigenin (DIG)- and dinitrophenol (DNP)-labeled primer, respectively, allowing specific binding to microparticles coated with the corresponding antibody. Both amplification products were biotinylated to enable fluorescence labeling with streptavidin-R-phycoerythrin. The mean fluorescence intensity of each microparticle population, corresponding to the amount of bound PCR product, was measured in a flow cytometer. We constructed microparticles coated with antibodies against DIG and DNP to specifically capture PCR products derived from target and competitor sequences, respectively. Results: As required for a reliable competitive PCR assay, nearly identical kinetics were found for the amplification of target and competitor sequences when using only one competitive primer. The method was applied to examine interleukin-8 expression in human lymphocytes after x-irradiation. One hour after irradiation, the concentration of transcripts decreased by half. Conclusions: The flow cytometric assay for the quantification of competitive RT-PCR products avoids additional hybridization steps and antibody labeling. The use of paramagnetic microparticles would also enable the complete automation of this method." @default.
• W2137060355 created "2016-06-24" @default.
• W2137060355 creator A5033808305 @default.
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• W2137060355 date "2002-09-01" @default.
• W2137060355 modified "2023-10-07" @default.
• W2137060355 title "Flow Cytometric Quantification of Competitive Reverse Transcription-PCR Products" @default.
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• W2137060355 doi "https://doi.org/10.1093/clinchem/48.9.1398" @default.
• W2137060355 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12194914" @default.
• W2137060355 hasPublicationYear "2002" @default.
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