| Popis: |
Determination of cell lineage in acute leukemias is essential for diagnosis and treatment. Detection of myeloperoxidase (MPO) mRNA establishes myeloid lineage of leukemic blasts that may be too primitive to be identified as myeloblasts based on morphology, cytochemistry, or immunophenotype. A highly specific and sensitive new procedure for MPO mRNA detection has been developed using HL-60 cells. It involves a microprocedure for total cellular RNA extraction, reverse transcription, and specific amplification of target sequences in the resulting MPO cDNA, by the polymerase chain reaction. Specific primers are designed to amplify an 89-base pair (bp) sequence from the signal peptide, 179 and 318-bp sequences from the start and end, respectively, of the heavy-chain sequence, and a 255-bp sequence overlapping the proregion and light chain. The correct-size amplification products, detected electrophoretically, demonstrate MPO mRNA expression in the leukemic cells analyzed. The sensitivity of this new procedure was evaluated on serial concentrations of HL-60 cells and was found to be 10-10(4) cells depending on the MPO cDNA amplified sequence. No amplification products were obtained using peripheral blood lymphocytes as a negative cellular control. The specificity of the procedure is demonstrated by Southern blotting and hybridization with 32P-labeled oligonucleotide probes specific for each of the amplified sequences. An additional advantage of this procedure is availability of results in 8-24 h, compared with 1-2 weeks for conventional RNA methods. |
