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Clinical and Diagnostic Laboratory Immunology, April 2005, p. 477-483, Vol. 12, No. 4
1071-412X/05/$08.00+0     doi:10.1128/CDLI.12.4.477-483.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Simplified Fluorescent Multiplex PCR Method for Evaluation of the T-Cell Receptor Vß-Chain Repertoire

Sanjit Fernandes, Surendra Chavan, Vivek Chitnis, Nina Kohn, and Savita Pahwa*

Immunology and Inflammation Center of Excellence, North Shore—Long Island Jewish Research Institute, North Shore University Hospital—NYU School of Medicine, Manhasset, NY 11030

Received 11 March 2004/ Returned for modification 20 July 2004/ Accepted 19 November 2004

Rationale: evaluation of the T-cell receptor (TCR) Vß-chain repertoire by PCR-based CDR3 length analysis allows fine resolution of the usage of the TCR Vß repertoire and is a sensitive tool to monitor changes in the T-cell compartment. A multiplex PCR method employing 24 labeled upstream Vß primers instead of the conventionally labeled downstream Cß primer is described. Method: RNA 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 Cß primer and an Omniscript RT kit. The 24 Vß primers were multiplexed based on compatibility and product sizes into seven reactions. cDNA was amplified using 24 Vß primers (labeled with tetrachloro-6-cardoxyfluorescein, 6-carboxyfluorescein, and hexachloro-6-carboxyfluorescein), an unlabeled Cß primer, and Taqgold polymerase. The fluorescent PCR products were resolved on an automated DNA sequencer and analyzed using the Genotyper 2.1 software. Results: Vß spectratypes of excellent resolution were obtained with RNA amounts of 250 ng using the labeled Vß primers. The resolution was superior to that obtained with the labeled Cß primer assay. Also the numbers of PCRs were reduced to 7 from the 12 required in the Cß labeling method, and the sample processing time was reduced by half. Conclusion: The method described for T-cell receptor Vß-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 Vß repertoire analyses in clinical trials.

* Corresponding author. Mailing address: University of Miami School of Medicine 712, Batchelor Children's Research Institute, University of Miami School of Medicine, 1580 N.W. 10th Avenue, Miami, FL 33136. Phone: (305) 243-7732. Fax: (305) 243-7211. E-mail: spahwa{at}med.miami.edu.

Clinical and Diagnostic Laboratory Immunology, April 2005, p. 477-483, Vol. 12, No. 4
1071-412X/05/$08.00+0     doi:10.1128/CDLI.12.4.477-483.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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