Quantification of TRECs
Genomic DNA was isolated from peripheral blood samples according to standard protocols (QIAGEN). The sequences of the primers used for PCR were as follows: TRECs, 5′-CACATCCCTTTCAACCATGCT-3′ and 5′-GCCAGCTGCAGGGTTTA GG-3′; probe, 5′-FAM-ACACCTCTG GTTTTTGTAAAGGTGCCCACT-3′-TAMRA; and T cell receptor alpha constant sequences, 5′-AGGAATCCTTGTCTCTGAAAAA TGC-3′ and 5′-TTCCTTTTAGTTTCTTGGCCTATGC-3′; probe, 5′-HEX-TGAAGA GAGGACCCTGTTACCGCC A-3′-TAMRA. TRECs were measured by real-time quantitative PCR, as described previously. Briefly, 5 µl of DNA was amplified in 25 µl of PCR solution containing primers and probes specific for TRECs. Standards were prepared according to protocols published by Sottini et al17. All DNA samples were run in triplicate alongside no-template controls.