We are recently trying to isolate RNA of FACS sorted cells with a population of 100K. The cell population we are sorting is very sensitive and we noticed a lot of cell death after the sorting procedure. For RNAisolation we are using RNeasy Mini Kit from Quiagen. Although we tryied to collect the cells with different conditions (PBS, RLT, BSA coated falcon), we did not manage to isolate any RNA. Our unsorted population however gives us a high RNA yield.
Has anybody had similar problems and has a solution we could try?
If you can, lightly aldehyde fix the cells and use a kit to isolate RNA.
It might require resuspending the cells, in PBS, and then lysing in Trizol. Followed by chloroform extraction, adding NaCl (final 250mM) to neutralize the RNA backbone, and 2.5 volumes of Ethanol for overnight precipitation.
Suspend the 100K cell pellet in 100 uL PBS, then add 1 mL of Trizol, proceed with usual Trizol RNA isolation procedure (be sure to recover at least 550 uL of the aqueous layer during the Trizol procedure), then, after the air-drying step of the Trizol RNA isolation procedure, resolubilize the final (non-visible) pellet in 10 uL of the nuclease-free water or buffer of your choice. This should get you some nice RNA concentrations.
100K cells (Kasumi1, K562 cell line in our case) give the yield about 2-3 mkg of RNA/ I used 0.5 ml of TRIZOL. Also, with sorted cells (K562) there were no problem with the yield in comparence to unsorted control cells. But I recomend to wash the cells with 1xPBS before isolation (resuspend cells in PBS 1 ml, centrifuge cells, add trizol)
From our experience, it is best to FAC sort the cells straight into Trizol-LS and perform RNA extraction using the basic phenol-chloroform based method. We also worked with very small population of cells. Using commercially available kits actually reduced our yield significantly (to practically nothing). You can also check out the protocol that we published.
Article Identification of differentially expressed genes during deve...
First of all, try to get some RNA from 100% of live cells you pass through the sorter. We sort a lot directly into RLT (don't forget 2ME to be added ex tempora) and 100K cells should be EASY. If you confirm that your sorted vs unsorted RNE preps yield similar results, re-evaluate your gating. I saw people gating on triple-negative populations and ending up sorting debris instead of cells.
Feel free to give us more details about your sort, it might get more clear then what the problem is about.
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