RT-PCR from Eosinophil-Depleted Leukocytes without RNA Extraction: Cell Selection Using Streptavidin PCR Tubes

Abstract

Objectives: The major RNase activity of leukocytes has been attributed to eosinophil-derived neurotoxin EDN. Depletion of eosinophils enables RT-PCR from 10 5 leukocytes without RNA extraction. In this study we introduced streptavidin-coated PCR tube strips for the selection of eosinophil-free leukocytes for RT-PCR analysis. Design and methods: Polypropylene 0.2 ml PCR tube strips were coated with streptavidin and biotinylated antibodies against cell surface antigens were attached to the tubes. CD7-positive T-lymphocytes, CD19-positive B-lymphocytes and CD16-positive cells (mainly neutrophils and monocytes) were positively selected by incubating of 1-2 × 10 5 leukocytes in the antibody-coated PCR tubes for 30 min at 23°C. Results: The mean amount of cells bound into a tube was 31,500 (CV25%) T-cells and 8,600 (CV61%) B-cells from 12 blood samples, and 23,600 (CV22%) CD16+ cells from 17 samples. The influence of selected cell lysate on the RT-PCR analysis of Philadelphia chromosome ( bcr/abl translocation) from 100 K562 cells was small: 78% (CV28%) of the leukocyte-free signal was obtained in the presence of CD16+ cells or 89% (CV15%) and 99% (CV11%) and in the presence of T-cells and B-cells, respectively. Conclusions: These results suggest that through the introduction of eosinophil-free cell population into RT-PCR a reproducible method with reasonable leukocyte yield and avoiding RNA extraction was developed.