RT-PCR for TRGV Subfamily Amplification and Genescan Analysis for TRGV Subfamily Clonality Analysis

Three sense TRGV primers and a single TRGC reverse primer were used in unlabeled PCR for the amplification of the TRGV subfamilies. Runoff PCR was performed with fluorescent primers labeled at the 5’ end with the FAM fluorophore (Cγ-FAM) (TIB MOLBIOL GmbH, Germany). A DNA thermal cycler (BioMetra, Germany) was used to perform this reaction process. The primers are listed in Table 2. PCR was performed as described in our previous report (19–21). Aliquots of cDNA (1 μL) were amplified in 20 μL reactions with one of the three Vγ primers and a Cγ primer. The final reaction mixture contained 0.5 μM sense primer and antisense primer, 0.1 mM dNTPs, 1.5 mM MgCl₂, 1× PCR buffer, and 1.25 U Taq polymerase (Promega, USA). After 3 min of denaturation at 94°C, 40 PCR cycles were carried out (94°C for 1 min, 60°C for 1 min, and 72°C for 1 min and a final elongation for 6 min at 72°C). All PCR products were stored at 4°C and ready for Genescan analysis (22).

Aliquots of the unlabeled PCR products (2 μL) were subjected to a cycle of runoff reaction with a fluorophore-labeled Cγ-FAM primer. The labeled runoff PCR products (2 μL) were heat-denatured at 94°C for 4 min with 9.5 μL of formamide (Hi-Di Formamide, ABI, USA) and 0.5 μL of size standards (GENESCAN™-500-LIZ™, Perkin Elmer, USA). The samples were then loaded on 3100 POP-4™ gel (Performance Optimized Polymer-4, ABI, USA) and resolved by electrophoresis in an ABI 3100 DNA sequencer for size and fluorescence intensity determination using Genescan software (23).