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

Abstract

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

Cytomegalovirus‐specific CD8+ T cells targeting different peptide/HLA combinations demonstrate varying T‐cell receptor diversity

“…These included optimized ratios of primer concentrations, which were multiplexed according to compatibility and amplicon size as listed in Table 2. A standard set of 24 Vβ primers that represent 22 TRBV subgroups 14,15 was used (modified from refs 16 and 17). Table 3 indicates the correspondence between the primer names based on the nomenclature by Wei et al.…”

HLA-Cw and TCR vβ analysis in twenty Mexican patients with psoriasis

“…The isolated RNA was reverse transcribed using oligo-dT primer and SuperScript II RNase H Reverse Transcriptase (Invitrogen). For amplification of the cDNAs, we have used the set of 24 pair of primers described by Fernandes et al [14]. The RT-PCR products were run in 2% agarose gel and stained with ethidium bromide.…”

“…The hypervariable region of the TCR Vβ gene family was examined as reported by several authors [10,13,14] ( Table 3). In a subset of regions within group 1, the Vβ2 and Vβ7 subfamilies were predominantly expressed.…”