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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!

-sorter-

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?).

-vairus-

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!).

-sorter-