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isolating RNA from FACS - (Oct/05/2006 )

I FACS-sorted stem cells directly into RNALater instead of buffer/serum since I want to minimize the time that stem cells may differentiate during a 2 and half hour sort. But I was unable to pellet cells using sequentially 1000g, 5000g, 10000g. Ambion's website suggested diluting RNALater with PBS 1:1 prior to spin to decrease the viscosity. I did that and spun at 10000g, still no pellet. Then I used Trizol Reagent at 10:1 volume (now I am up to 30 ml), and even after adding glycogen at the isoprop precip step, I still had no pellet!!! Does anyone have any suggestions for my future RNA harvest from stem cells out of the FACS? THANKS!


How many cells did you sort? Did you check under a microscope if you still have cells? (if you have too little cells, you wont see the pellet maybe?).


I sorted half of a million cells in the tube. On a previous run, I sorted into PBS/serum and was able to successfully pellet at 450g. I did not check under the microscope this time, but I have previously (besides, this time, there was no pellet!).