Hello,
we are using anti CD45+TER119 magnetic beads in order to get a clean endothelial cells fraction. we tried various protocols for RNA isolation, and we think that there might be a problem with the FACS protocol itself.
looking for a new way to isolate endothelial cells and get good RIN.
How do you control the quality of your RNA? And which RNA are you looking for? 300,000 cells are not so much...
I'm not looking for a specific type, we want to know the changes in RNA expression profile due to VEGF induction .
these cells should be enough , I know it from other labs next door. it shouldn't be so hard to separate the endothelial cells , something with our protocol might be the problem.
I assume you are using CD45+TER119 approach for indirect isolation of EC. In this case a single CD45+TER119-based EC isolation approach may not be enough to get high purity EC population (CD45+TER119 only removes leukocytes from the cell suspension). Have you considered to use anti-CD31 (PECAM-1) beads?
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