From: Al Sabirsh (alan@biogen.wblab.lu.se)
Date: Wed Oct 30 1996 - 06:25:11 EST
Hello to all, We have cloned a receptor that we wish to detect in peripheral blood cells using fluorescent in situ hybridization and flow cytometry. Originally, I had planned to use RT-PCR in situ, but the open reading frame for this receptor (where our primers are located) consists of only one exon and thus the primers bind to genomic DNA and contaminating bands are produced. We do not as yet know enough about the genomic arrangement of the receptor to accurately predict where any useful introns could be. So I am now going to use a fluorescently marked riboprobe to label the relevent mRNA and hope that the mRNA signal will be large enough to distinguish cells actively producing this receptor from background genomic binding. (A riboprobe is being used because unbound probe can be degraded, facilitating diffusion out of the cells and thus lowering background). I was wondering if anybody had a better suggestion. If our cells are not producing much message we are not going to detect anything, so I would be grateful if someone can suggest a better way to do this. (Note: the fluorescence in situ hybridization is being used together with flow cytometric immuno-phenotyping. Northerns work fine, but don't give us information about the cell population expressing the receptor). Al Sabirsh Molecular Neurobiology Lund University Sweden alan@biogen.wblab.lu.se
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