We'd like to extract genomic DNA from the cells leftover at the bottom of the tube after a Lymphoprep PBMC isolation, rather than waste any whole blood to extract DNA (as we want to maximise the PBMC yield). Does anyone have experience with this? Do the cells need a lot of wash steps to remove residual Lymphoprep?
Hi Ceri Roberts
I am not sure that are you kidding every one in Research Gate, Since you know that RBC lacks their nucleus and mitochondria while they get matured, then where goes to find the DNA. Even if you dose not know about that its fine then just read in below link
https://en.wikipedia.org/wiki/Red_blood_cell
When high quality and quantity of DNA could be isolated from WBC, why to go for RBC? Yes to lyse RBC, sometimes it requires more wash steps but it depends on the RBC count of the sample. Refine your protocol, you could get high quantity of DNA from WBC itself !
Thank you for your responses. I have been told that one can extract DNA from the erythrocyte/granulocyte layer (the red layer) leftover at the bottom of the gradient (you are quite right about RBC, Sundarajan, so I suppose that this DNA would be extracted just from the granulocytes). We would like to maximise the number of PBMC we can get out of the blood for other experiments, so using the bottom layer for this would be ideal as it is usually discarded. Any advice from those who have tried it would be much appreciated.
Hi Ceri Roberts
If you need to have more PBMC for experiments (even for DNA isolation) you can treat your cells with PHA for 48 hr.
Also in order to separate granulocytes you can use Histopaque 1077 and 1119 has an combined gradient where in the RBC will be completely settled down while granulocytes and other lymphocytes will separate according to their gradient.
All the Best
As I said, we use the PBMC (for functional experiments, so PHA stim would not be appropriate); my question is specifically about isolating DNA from the bottom of a Ficoll gradient. I know that this can be done but would like protocol advice from anyone who has tried it.
It's presumably too late now, but in case anyone else stumbles across this while looking for something else as I did, this paper might be useful: http://www.biomedcentral.com/1756-0500/6/440
Hi Ceri, have you tried this protocol? I´m planning to do the same, my PBMC are very precious and I cannot use PHA for my experiments. So, I want to isolate DNA from granulocyetes.
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