A simple subtraction method for the isolation of cell-specific genes using magnetic monodisperse polymer particles.
In this study we present a simple subtraction method for the isolation of cell-type-specific genes using magnetic beads. Biotinylated first-strand cDNA is generated from one cell type and immobilized onto magnetic streptavidin beads. Poly A+ RNA, isolated from a different cell type by use of oligo-dT beads, is then hybridized to the immobilized cDNA. Beads with hybridized mRNA are subsequently removed from the solution by attraction to a magnet. The cell-specific mRNA, left in solution, is finally converted to a radiolabeled cDNA probe in order to screen cDNA libraries. In this study, we present an example of a successful subtraction strategy involving three cell types: the pre-B-cell line Reh; 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated Burkitt's lymphoma line Daudi; and TPA-stimulated T lymphocytes. This strategy was chosen due to our interest in gene products known to be expressed in TPA-stimulated B and T lymphocytes, but not in Reh cells. Several previously unknown genes were identified.
Institute for Cancer Research, Norwegian Radium Hospital, Oslo.
H C
HC
Aasheim
A
A
Deggerdal
E B
EB
Smeland
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E
Hornes
D001402:B-Lymphocytes / Q000737:chemistry; D015152:Blotting, Northern; D015139:Blotting, Southern; D015342:DNA Probes; D018076:DNA, Complementary / Q000302:isolation & purification*; D006801:Humans; D008280:Magnetics; D008863:Microspheres; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013601:T-Lymphocytes / Q000737:chemistry; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D018076:DNA, Complementary; D012333:RNA, Messenger; D013755:Tetradecanoylphorbol Acetate