I work with intestinal epithelial cells(IECs) and would like to perform microarray analysis on freshly isolated (pure population of) primary IECs without any(or very little) cd45 population.
The following is my usual protocol -
1. Isolate IECs
2. Add CD45 AB(1 hour primary incubation) and use Dynabeads)anti-cd45 30mins incubation) for CD45 depletion.
3. Spin down the cells after depletion and suspend the cell pellet in RNA Later reagent for 24hours.
4. Proceed with RNA Isolation using the Qiagen Kit
Problems -
1.Very low RNA yield
2.RIN = 4-4.5
Any help on increasing RNA yield and quality of IECs post depletion would be highly appreciated.
Thanks
Antibody selection of live cells can drive cell death pathways. Try fixing your cells with 1% formalin for 5 minutes before immuno-selection.
Hi Michael,
Thanks for the response.
Would fixing prior selection in any way interfere with the RNA quality?
In my opinion, lightly fixing the RNA should not effect quality but be sure to control the time. Many researchers PCR from paraffin embedded tissue. Any other thoughts out there?
Hi Tharini,
If not, maybe performing every single step at 4° can increase your RIN.
Good luck!
Hi Sami,
Thanks for taking time out to reply. I do perform all the steps at 4deg.
Tharini
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