Hi researchers,
I was wondering if anyone knows a way I could Isolate RNA and Protein from the same cell pellet. I'm not looking for a kit that allows me to do both at the same time unless that is my only option. Basically I have cell pellets I have stored at -80 degrees that range from 800,000-9x10^6 cells per pellet. Because I really only need about 100,000 cells for RNA isolation for qPCR I dont want to waste an entire pellet of cells on just RNA. I need several million cells to achieve enough protein for my western blot analysis. So is there a way I could resuspend this pellet and potentially use like 20% of the lysate/resuspension for RNA and the other 80% for western blot analysis at a later time without damaging the cells or compromising one procedure for another? My big concern is the lysing of the pellet with one type of buffer/breaking the cells and it compromise either of the procedures...
Thanks!
Andrew Lewis
Thanks! I was thinking the same thing but I was hoping maybe someone had a trick I haven't seen yet. I think my best bet would be dividing the pellet into separate smaller pellets before freezing them down them like you said, thanks for your help!
I'd recommend splitting before freezing in the future, but I think you should be fine to split the cells for each assay after thawing them out.
I'd recommend RNAble or TriReagent which separates both RNA and proteins.
or
If you use RNeasy kit for the RNA extraction, there is a complementary protocol to extract protein from the first discarded flow-through. That can be another solution to your problem
You can use TRIzol method, where you can easily separate RNA, DNA and protein after addition of chloroform. The RNA will be in aqueous solution, you take it out first and further separate your whole Protein. Dissolve your respective pellets in TRIzol reagent.
Hi Haley,
Qiagen has several kits that may work well for you. I have not used them with frozen cells personally, but have pull RNA and Protein from freshly collected cells and from frozen tissue. I would consult the data sheets and/or tech support to make sure that you wouldn’t have interference with your downstream assays.
Hi Haley,
Qiagen has several kits that may work well for you. I have not used them with frozen cells personally, but have pull RNA and Protein from freshly collected cells and from frozen tissue. I would consult the data sheets and/or tech support to make sure that you wouldn’t have interference with your downstream assays.
Hi Haley,
Probably the trizol reagent is the best option for your problem. You can separate RNA and protein without divide pellet or damage cells.
Here, you can find a protocol of extraction with trizol reagent.