RT-PCR analysis showing MLL-AF-4 chimeric mRNA. Initial and second round RT-PCR reactions were performed using random hexamer-primed cDNA prepared from total RNA from the leukemic cells of patient 38. Sequences of sense MLL primer from exon 6 and LTG4AS2 antisense primer from AF-4 have been described.18 Four separate initial and second round RT-PCR reactions each gave a single 741-bp product, as shown by example in the figure (top). Direct sequencing of the products of each second round reaction showed an in-frame fusion of MLL exon 8 to AF-4 at position 1459 in AF-4 cDNA19 (bottom). The predicted 627-bp product was obtained in positive control cell line RS4:11 (top). RNA negative controls (dH2O) for initial and second round reactions also are included (top).
