Hello,
I am sorting monocytes from spleen for RNAseq. I pre-purify my cells using Miltenyi isolation kits and then I FACS sort the cells to obtain a high purity. Since I had problems with the RIN values (below 8) I added RNAse inhibitors to the sorting buffer and the recollection medium since the spleen contains a lot of RNAses. After this I increased the integrity of my RNA to reach values over 8 but not in all samples. Also I directly isolate the RNA after the sorting.
Does anybody knows how to further improve the quality of my samples? is the perfusion of the mouse with PBS with RNAse inhibitor a good option? it would be toxic for the cells?
Thank you in advance for your help.
Thank you for your answer.
The viability of my cells is really high (>98%) since I add a viability dye to sort only those cells that are alive. However may some still die during the steps after since when I added RNAse inhibitors to the recollecting buffer I much improved the integrity of my RNA. My main issue is that still it is not a reproducible method since some samples have a RIN of 6-7 which is not suitable for RNAseq while others are above 8.
You are right, Adding RNAse inhibitor to the perfusion buffer may affect the expression of my surface markers. Do you have any experience with that?
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