Molecular measurement of t cell receptor excision circles
This chapter provides protocols necessary for quantifying human, mouse, and nonhuman primate signal joint T cell receptor excision circles (sjTRECs) produced during T cell receptor alpha (TCRA) gene rearrangement. These non-replicated episomal circles of DNA are generated by the recombination process used to produce antigen-speci fi c T cell receptors. The number of sjTRECs per mg of thymus tissue or per 100,000 lysed cells has been shown to be a molecular marker of thymopoiesis and naïve T cells. This technology is bene fi cial to investigators interested in quantitating the level of naïve T cell production occurring under various circumstances in a variety of systems, and complements traditional phenotypic analyses of thymopoiesis. This chapter speci fi cally describes procedures required for rapid detection and quantitation of sjTRECs in thymus tissue or isolated cells using real-time quantitative polymerase chain reaction (PCR). The sjTREC assay system comprises species-speci fi c forward and reverse primers for ampli fi cation of a unique site on the T cell receptor δ( TCRD ) sjTREC, a fl uorescently labeled (FAM/ZEN/IABkFQ) species-speci fi c real-time probe, and a species-speci fi c sjTREC DNA plasmid standard for quantitation. © Springer Science+Business Media New York 2013.
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