Establishment of the real-time fluorescence quantitative reverse transcription polymerase chain reaction with DNA melting curve analysis for detecting the CDR3 skewing of TCR beta gene repertoire in the human peripheral blood
MA Rui
LUO Rong
SUN Wanbang
YAO Xinsheng
Abstract：Objective To establish the technique of the real-time fluorescence quantitative reverse transcription polymerase chain reaction(FQ-PCR) with DNA melting curve analysis for detecting the CDR3 skewing of TCR beta gene repertoire in the human peripheral blood. Methods The total RNA of peripheral blood mononuclear cell (PBMC) from 4 healthy donors and 9 colorectal cancer patients were transcripted reversely into cDNA. The cDNA of 26 TRBV gene family CDR3 was amplified by the FQ-PCR ; the monoclonal/oligoclonal/polyclonal CDR3 spectratyping was analyzed with DNA melting curve. Results The FQ-PCR products of 26 TRBV families CDR3 showed a blur land in the prediction of products size in healthy donors, while parts of TRBV family CDR3 prod-ucts disappeared in patients on 1.5% agarose gel by Gold - View staining. The 26 TRBV families CDR3 showed different frequency by relative fluorescence quantitative detection in healthy donors and patients. The CDR3 spectratyping of 26 TRBV families showed poly-clonal peak ( Gaussian distribution) in healthy donors but showed different monoclonal/oligoclonal/polyclonal peak in the colorectal cancer patients with DNA melting curve analysis, namely melting curve spectratyping of CDR3'. Conclusion The study suggests the technique of FQ-PCR with DNA melting curve analysis'is convenience and celerity method for detecting the CDR3 skewing of TCR beta gene repertoire in the human peripheral blood.
Keywords：T cells receptorcomplementarity determining region 3real-time fluorescence quantitative reverse transcription polymerase chain reactionmelting curve
Publication Date：2009-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：6( 446-451 )