Hello, I have been trying to extract RNA from about 1.5x10^5 freshly isolated human PBMCs with the Qiagen Rneasy Mini kit. The fresh blood is layered on top of Cedarlane lympholyte in a falcon and then centrifuged to obtain a buffy coat. After that I collected the buffy coat, washed and counted the cells and resuspended in complete RPMI. Then, I put about 1.5x10^5 cells in single wells in a 96-well cell culture plate and stimulated half of the wells with pokeweed mitogen. The cells were incubated for an hour at 37 degrees and 5% CO2, and then I pelleted the cells and tried isolating RNA with the Rneasy mini kit. However the yield was very low. I tried isolating RNA from PBMCs which were not stimulated and just resuspended in RPMI in eppendorfs and the yield was better but not very good either.
I am also trying to stimulate fresh whole blood with pokeweed mitogen, and will be trying with tetanus toxoid later. About 100 uL of blood is put in wells on a 96 well plate and incubated for an hour and then RNA isolated, but again with low yield.
Is just a question of the low blood volume or does anybody know how to improve the yield using such a small volume? Do you think I am not pelleting the PBMCs in the first example properly and taking the supernatant off so that's interfering with the yield?
Any help will be very much appreciated.
Hi Anna,
To me the low blood volume seems to be the prime factor for the low yield of RNA. Can you please try to double the volume of your starting blood sample with equal volume of PBS and then try to get the buffy coat. It might help you to reduce the loss of PBMCs from well formed buffy coat. Thanks
Low RNA yield seems to be related to your smal blood volume and low cell number used.
Try to use at least 10`6 celle per 6 well plates, I had more RNA using 6 well plates.
Good luck
Hi Anna,
I have uploaded my favorite protocol for RNA extraction. If you cannot increase your starting cell numbers, then you might try to use an RNA carrier, such as glycogen between steps 2 & 3 of this protocol.
Good Luck!
Kristi
I like Kristi's answer.
When I isolated PBMCs from RBC-depleted blood I obtained low no. of PBMCs. but still my starting blood volume was ten times more than that of Anna, so,I did not have to use any RNA carrier.
Thank you Kirsti, for the moment I am stuck with Qiagen Rneasy minikit, and my supervisor wants me to use these small volumes. However, I might try your advice if we cannot increase the starting volume! Thank you.
Anna
Yes, Anna, I understand. Also note that my protocol uses the Qiagen RNeasy minikit. It just has a modification to the protocol that requires chloroform and Trizol. You may already have those reagents in your lab, and if not, they are inexpensive.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques