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.