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I will soon be sorting cells to obtain RNA from sorted cell populations. I
was wondering if anyone had dealt with such issues as rinsing the lines with
RNAse inhibitors, sorting directly into solutions containing RNAse
inhibitors, keeping the instrument cold, etc. We use a BD FACStar Plus. I
believe Peter Openshaw from St. Mary's Medical School asked essentially the
same question 4 months ago, with more emphasis on somehow preserving the
cells for post-sort analysis. I do not yet know how imperative that issue
will be for this project but any suggestions or insights would be gratefully
appreciated. Please send replies to tstaley@amgen.com.
Tabitha Staley
Amgen, Inc.
Thousand Oaks, CA
FAX (805) 447-1982
TEL (805) 447-1196
In response to your question about ultimately extracting RNA from sorted
cells, I have had much experience. My PhD thesis project involved sorting
cells at specific points within the cell cycle, based upon hoechst 33342
quenching from BrdU incorporation, and extracting RNA for quantitative
purposes. In the beginning I attempted to keep everything sterile, i.e. run
ethanol through the lines, swab down the workstation with ethanol and keep
everything cold...as time went on and I realized that I may be a graduate
student forever, I decided to save some time and see what would happen if I
bagged sterility. I sorted my cells (murine macrophages) into
"relatively" cold medium (the sorts were often long and the medium warmed up)
containing antibiodics and 10% FCS (as a cushion) without doing any sterile
preparation of the FACStar Plus. After sorting I counted the cells, and
immediately continued with an RNA extraction protocol. I found that the
cells remained intact throughout the sort, there was no problem with RNAse
contamination and I could easily sort and extract intact RNA in one day from
numerous time points (of course that is based upon graduate-student time).
So my advice is try doing a run-of-the mill nonsterile sort into cold medium
before you decide to gown up and do your experiment in a plastic bubble!
PS: My mentor, Carleton Stewart, might not like to know that I cut corners
to finish my PhD project...though he may have been glad to get me out of the
lab!!
good luck,
M.C. Riedy
M.C. Riedy
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