I need a cell that will be HLA-DR positive on RT-PCR. For this, would it work if I directly isolate RNA from PBMC cells with Ficoll? Or should I necessarily culture these PBMCs and isolate T cells from there?
I suggest you use fluorescent anti HLA-DR antibodies and sort the cells from your samples using a cell-sorter (flow-cytometer). you can directly sort the cells into lysis buffer which will protect the RNA from degradation. Make sure to block you cells first to prevent non-specific binding. Then proceed with cDA synthesis and qPCR as usual.
Highly agree that FACS is the best way to isolate and enrich your target population. However, if such a technique is unavailable or too expensive, you can try an alternative way of enriching your population.
PBMC are comprised mainly of lymphocytes and monocytes. If you want to get rid of the monocytes, a short-term culture of your PBMC should make the monocytes adhere to the plastic so the culture suspension will be monocyte-free (not 100%, but the vast majority of monocytes will be gone).
This is nothing compared to FACS, but i just wanted to add it for the record as a more "primitive" way of enriching lymphocyte population. Sometimes sorting capable flow cytometers or expensive antibodies are not around.
Also, anti-CD3 is a very commonly used antibody. There's probably no lab without a vial or two. If this is available, sorting only CD3+ cells is another alternative to enrich T-cell populations. Not as specific as a HLA-DR sorting given your particular interest, though.
In PBMCs, B cells and Monocytes are HLA-DR positive, while small number of T cells are positive for HLA-DR.
So, if you want to extract RNA from HLA-DR positive T cells then sorting (FACS or MACS) is must.
Or if you want just HLA-DR positive cells then go ahead with PBMC.
- Badges
- Science method