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Spleen homogonization for RNA extraction issue - (Oct/14/2013 )

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?


That gloop is probably genomic DNA.  (mammalian) RBC do not contain DNA so the kit will not contain any DNases.  Try using a tissue RNA extraction kit.