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
- - - - -

Sequencing virus from liver samples


  • Please log in to reply
1 reply to this topic

#1 liguerr

liguerr

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 06 April 2018 - 07:28 AM

We have long been baffled by trying to sequence RNA virus from infected animal liver. The ct is always fairly low (18-25) but when we try to perform RtPCr, it's almost impossible and through NGS (RNAseq shotugun random primers), there are virtually zero reads mapping to virus. We even did experiments in which we mixed a virus isolate of known titer with a liver sample bought from a grocery store, then compared to the virus isolate alone, and found the liver destroyed the RNA. It is assumed that enzymes in the liver are affecting RNA quality. Has anyone found a technique or some sort of extraction reagent or technique that might solve this issue? Thanks in advance!



#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 6,482 posts
536
Excellent

Posted 06 April 2018 - 08:10 AM

Liver is a known problem for its high level of RNases. The best bet that I know of is to perform old-school trizol style extractions, taking extra care to not carry over any of the protein layer. RNase inhibitors help a little, but you need to use more than you would for other tissues. Repeating extractions on samples can also help, though you tend to lose a bit of RNA this way.

 

You will also need to ensure that your RNA is aliqoted and stored in single-use aliqots at -80.  






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

©1999-2013 Protocol Online, All rights reserved.