Expression of the chimericHIP1/PDGFβR mRNA in patient bone marrow. RT-PCR analysis of HIP1/PDGFβR was performed using total RNA (2 μg) from t(5;7) patient bone marrow that had been reverse transcribed using the Qt primer. PCR was performed using a HIP301F forward primer and the PDGFβR 1848R primer for 30 cycles (94°C for 1 minute, 60°C for 2 minutes, and 72°C for 3 minutes). Nested PCR was performed on PCR products from the first reaction diluted 20-fold and amplified using the same PCR reaction conditions with HIP1 forward primers HIP721F, HIP1141F, HIP1561F, HIP2372F, HIP2494F, and HIP2613F in lanes 1 through 6, respectively, and the PDGFβR reverse primer 1806R. HIP1 primer numbers correspond to the first nucleotide of a 20-bp primer of HIP1 sequence according to Kalchman et al.20 The expected band sizes are 2,279, 1,859, 1,439, 628, 506, and 387 bp for lanes 1 through 6, respectively. Control experiments with no template or in the absence of reverse transcriptase gave no PCR product (not shown). TheHIP1/PDGFβR fusion was not detected in normal bone marrow and neither was the reciprocalPDGFβR/HIP1 fusion transcript detected in t(5;7) patient bone marrow using a nested PCR reaction with primers PDGFβR1691F, PDGFβR1711F, HIP13071R, and HIP12966R (primer numbers correspond to the first nucleotide of a 20-bp primer of either PDGFβR22 orHIP120 sequence).
