I plan to sort peripheral blood neutrophils from septic mice to determine differential gene expression between groups. The sorting will be done on a BD ARIA II flow sorter. How are cells typically sorted for use in gene arrays? Sorting into trizol, RNAlater, or what? Are any special precautions recommended when sorting for gene expression?
I would suggest RNA Later. The viability of cells can be affected during the sort process with non-viable cells susceptible to nucleases. Furthermore, RNA Later can be used at room temperature without any needs for specialised hoods (Trizol) or conditions.
A simple experiment would be to compare in-house differing solutions and their overall affect on RNA by measuring the RIN. repeat a couple of times for confirmation. Lengthy yes, but that should answer your question (hopefully!).
I have performed this experiment and compared circulating neutrophils in the nieve mouse and compared it to inflammatory neutrophils. ARIA sorting into PBS on ice followed by lysis in RNA extraction buffer with b-mechap worked well for signals that should be transient (eg COX2).
I have successfully sorted lymphocytes into medium or PBS with serum and extracted RNA from the cell pellet using the Trizol method. The RNA was then successfully used for real-time PCR.
I have no experience with sorting neutrophils, but a correctly working cell sorter should give you high viability sorted cells if the input cells are healthy. Nevertheless it's always best to confirm the viability of the sorted cells prior to use in downstream assays such as the one you describe.
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