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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
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