ArticleClinical and diagnostic laboratory immunology2005
Simplified fluorescent multiplex PCR method for evaluation of the T-cell receptor V beta-chain repertoire.
Article in Clinical and diagnostic laboratory immunology, 2005. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 2 papers.
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5 authors.
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Abstract
rationaleEvaluation of the T-cell receptor (TCR) V beta-chain repertoire by PCR-based CDR3 length analysis allows fine resolution of the usage of the TCR V beta repertoire and is a sensitive tool to monitor changes in the T-cell compartment. A multiplex PCR method employing 24 labeled upstream V beta primers instead of the conventionally labeled downstream C beta primer is described.
methodRNA was isolated from purified CD4 and CD8 T-cell subsets from umbilical cord blood and clinical samples using TRI reagent followed by reverse transcription using a C beta primer and an Omniscript RT kit. The 24 V beta primers were multiplexed based on compatibility and product sizes into seven reactions. cDNA was amplified using 24 V beta primers (labeled with tetrachloro-6-cardoxyfluorescein, 6-carboxyfluorescein, and hexachloro-6-carboxyfluorescein), an unlabeled C beta primer, and Taqgold polymerase. The fluorescent PCR products were resolved on an automated DNA sequencer and analyzed using the Genotyper 2.1 software.
resultsV beta spectratypes of excellent resolution were obtained with RNA amounts of 250 ng using the labeled V beta primers. The resolution was superior to that obtained with the labeled C beta primer assay. Also the numbers of PCRs were reduced to 7 from the 12 required in the C beta labeling method, and the sample processing time was reduced by half.
conclusionThe method described for T-cell receptor V beta-chain repertoire analysis eliminates tedious dilutions and results in superior resolution with small amounts of RNA. The fast throughput makes this method suitable for automation and offers the feasibility to perform TCR V beta repertoire analyses in clinical trials.
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