Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Spleen homogonization for RNA extraction issue


  • Please log in to reply
1 reply to this topic

#1 RoeTS

RoeTS

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 14 October 2013 - 02:14 AM

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?



#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,830 posts
414
Excellent

Posted 14 October 2013 - 01:39 PM

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.






Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.