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
- - - - -

Low RNA yield after extraction by RNA blood mini kit.


  • Please log in to reply
2 replies to this topic

#1 PopBCT

PopBCT

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 21 December 2009 - 07:27 PM

Hi,
I have stuck in a process of RNA isolation from blood.

I would like to extract the RNA from rare cells in peripheral blood that should be enriched before extraction step right? Method that I've used is density gradient centrifugation by using Histopaque 1077. I started from 10 mL blood sample, followed by the Histopaque's instruction and finally I got large amount of cell pellete. Then I extracted RNA by QIAamp RNA blood mini kit. While I was adding the RLT buffer, the mixture was very viscous and many foam occurred. According to the instruction, additional RLT buffer is recommended and need another column. I decided to use one column and didn't add any RLT buffer then transfer to the spin column. So, I noticed that the volume of the lysate (after centrifuged) was lesser than the volume of added RLT buffer. Then I checked for RNA concentration by nanodrop spectrophotometer and the result show the low yield of RNA when comparing RNA that was isolated from 1mL of whole blood.

What should I do? Any suggestion?

Thank you

#2 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,112 posts
59
Excellent

Posted 21 December 2009 - 08:45 PM

usually column based kits from Qiagen give low yields, that's for sure, although the purity is high.

I extract RNA with Qiagen's RNA Mini from cells and it always gives me lower yield compared to Trizol method.

#3 PopBCT

PopBCT

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 21 December 2009 - 11:50 PM

usually column based kits from Qiagen give low yields, that's for sure, although the purity is high.

I extract RNA with Qiagen's RNA Mini from cells and it always gives me lower yield compared to Trizol method.


That's true about Qiagen's kit gives low yield but its column capacity is not full yet. According to manual, they claim that the column capacity is 100 microgram but I got RNA concentration just around 8-30 microgram. It should be more than that.




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

©1999-2013 Protocol Online, All rights reserved.