Detection of Vα14Jα281 transcripts with RT-PCR in inductive cultures.

Total RNA was extracted from the thymic lobes (RAG-1^−/−in C57BL/6 background) alone (lane 1) or lobes with sorted NK1.1⁺ TCRαβ⁻ cells in the presence (lane 3) or absence (lane 2) of PMA plus ionomycin, and RT-PCR was performed as described in “Materials and methods” with primer pairs Vα14 Leader/Cα-rev1 and Vα14/Jα281, RAG-1 5′/3′ and RAG-1 5′ nest/3′ nest, or EF-1α 5′/EF-1α 3′ for positive control. RT-PCR was also performed without RNA (lane 4) as control. Results are representative of 3 separate experiments.