Hi everyone. Hopeing someone could provide some advice for me! I'm trying to prepare murine spleen for RNA extraction using a Qiagen RNeasy kit. I'm having issues as my spleen suspension is not eluting completely through the spin colums after centrifugation- about half remains in the filter column! My protocol is below.
I mashed murine spleen in a cell strainer using the end of a plunger and rinsed these cells through with DMEM 10% FCS. After spinning this down, I resuspended the pellet in RBC lysis buffer and left for a few minutes. Topped this up with media and respun and resuspended the pellet in 600 ul of the Buffer RLT (with B-ME as per instructions).
I noticed at this point the suspension has brown bits in it, and its very gloopy. After resuspending 300 ul of this in an equal volume of 70% EtOH it's still very gloopy- hard to suck up into the pipette. I added this gloopy mixture to the spin colums and spun them down. However not all the suspension eluted through and about half was still in the filter column - seems to be blocking the filter! Can anyone give me any advice on how to avoid this from happening?