Nucleic Acids Research Advance Access published online on October 3, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn634
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Methods Online |
The application of real-time PCR to the analysis of T cell repertoires
1Department of Immunology, 2Department of Surgery and 3Department of Health Sciences Research, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
*To whom correspondence should be addressed. Tel: +1 507 284 9654; Fax: +1 507 284 3757; Email: wettstein.peter{at}mayo.edu
Received June 5, 2008. Revised September 4, 2008. Accepted September 6, 2008.
The diversity of T-cell populations is determined by the spectrum of antigen-specific T-cell receptors (TCRs) that are heterodimers of 
and β subunits encoded by rearranged combinations of variable (AV and BV), joining (AJ and BJ), and constant region genes (AC and BC). We have developed a novel approach for analysis of β transcript diversity in mice with a real-time PCR-based method that uses a matrix of BV- and BJ-specific primers to amplify 240 distinct BV–BJ combinations. Defined endpoints (Ct values) and dissociation curves are generated for each BV–BJ combination and the Ct values are consolidated in a matrix that characterizes the β transcript diversity of each RNA sample. Relative diversities of BV–BJ combinations in individual RNA samples are further described by estimates of scaled entropy. A skin allograft system was used to demonstrate that dissection of repertoires into 240 BV–BJ combinations increases efficiency of identifying and sequencing β transcripts that are overrepresented at inflammatory sites. These BV–BJ matrices should generate greater investigation in laboratory and clinical settings due to increased throughput, resolution and identification of overrepresented TCR transcripts.