Transcription of AML1/ETO in adult bone marrow from individuals without AML.

In the upper panel the 185-bp amplification product of the nested reverse trancriptase (RT)–PCR is shown in the positive control cell lineage Kasumi-1 (K) and 6 bone marrow samples of individuals without AML followed by the primary and nested negative controls (P, N, respectively). In the lower panel, the corresponding internal positive controls using an abl RT-PCR are shown. We used 2% to 3% of the total cDNA of each sample as PCR template. Goettingen primary PCR, AML1-A 5′-ACC TCAGGTTTGTCGGTCG-3′ (bp 1976 to 1974), ETO-B 5′-GAACTGGTTCTTGGAGCTCCT-3′ (bp 2211 to 2231). Goettingen nested PCR, AML1-C 5′-AAA AGCTTCACTCTGACCATCA-3′ (bp 2008 to 2029), ETO-D 5′-GGCATTGTTGGAGGAGTCAG-3′ (bp 2173 to 2192). Vienna primary PCR, AML1-1 5′-AGCCATGAAGAACCAGG-3′ (bp 1941 to 1960), ETO-1 5′-AGGCTGTAGGAGAATGG-3′ (bp 2265 to 2281). Vienna nested PCR, AML1-2 5′-TACCACAGAGCCATCAAA-3′ (bp 2062 to 2079), ETO-2 5′-GTTGTCGGTGTAAATGAA-3′ (bp 2229 to 2246). Hannover real-time PCR was performed as described.7 All nucleotide sequences refer to Miyoshi et al.8 An abl RT-PCR served as internal positive control and specificity of amplification was confirmed by cycle sequencing as described.4 The sensitivity of the AML1/ETO RT-PCRs was 10⁻⁶ (cell-in-cell dilution of Kasumi-1 in HL60). RNA preparation, reverse transcription, and PCR were performed in separate laboratories and in repetitive negative controls contaminations were not observed.