Detection of mRNA in flow-sorted cells.
Cells were sorted onto nitrocellulose filters which were saturated with a lysing cocktail designed to preferentially immobilize cellular mRNA. After washing, these filters were incubated with 32P-labeled specific DNA probes. We used the phorbol ester/lipopolysaccharide (PMA + LPS) co-induction of IL-1 mRNA and CD13 expression in U937 cells to demonstrate the specificity of the technique. In addition we used the abundant expression of c-fos in U937 to demonstrate linearity. IL-1 beta mRNA is readily discernable autoradiographically from as few as 5,000 PMA + LPS-induced cells sorted onto a filter. With liquid scintillation counting we demonstrate good linearity of the c-fos quantitation over the range of 1,000 cells to 60,000 cells per filter target. The technique is easily adapted to any sorting flow cytometer and should prove useful to help correlate any flow cytometric cell phenotype with specific mRNA abundance.
Dunne
J F
JF
Department of Immunology, Syntex Research, Palo Alto, California 94304.
Thomas
J
J
Lee
S
S
D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism; D016403:Lymphoma, Large B-Cell, Diffuse; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger / Q000032:analysis*
D007375:Interleukin-1; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger