RT-PCR from eosinophil-depleted leukocytes without RNA extraction: cell selection using streptavidin PCR tubes.
OBJECTIVE
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.


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 x 10(5) leukocytes in the antibody-coated PCR tubes for 30 min at 23 degrees 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.
Hämäläinen
M M
MM
Department of Clinical Chemistry, Turku University Central Hospital, FIN-20520, Turku, Finland. mauham@utu.fi
Eskola
J U
JU
Hellman
J
J
Pulkki
K
K
Rajamäki
A
A
D000293:Adolescent; D000328:Adult; D000368:Aged; D018941:Antigens, CD19 / Q000032:analysis; D019011:Antigens, CD7 / Q000032:analysis; D002469:Cell Separation; D004804:Eosinophils; D005260:Female; D006801:Humans; D007694:Killer Cells, Natural; D007962:Leukocytes; D008297:Male; D008875:Middle Aged; D010677:Philadelphia Chromosome; D012313:RNA / Q000302:isolation & purification*; D017452:Receptors, IgG / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019809:Streptavidin; D014407:Tumor Cells, Cultured
D018941:Antigens, CD19; D019011:Antigens, CD7; D017452:Receptors, IgG; D012313:RNA; D019809:Streptavidin