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.