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
- - - - -

Ct value very high. What can I do to improve this ct?


  • Please log in to reply
4 replies to this topic

#1 TV2013

TV2013

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 07 February 2013 - 07:26 AM

Hi everybody

I work with Sybr to analyze 5-HT (Serotonin receptor) in rat hypothalamus. However, during the primer optimization the ct value for this gene has been very high (30- 32). We have already redesigned twice the primers for 5-HT and we got same result. Last experiment, we concentrated the cDNA pool samples for 1:5 and the ct didn't change.
I don't know what to do in this case.
Can anybody help me?
Thanks a lot

#2 Mimoza

Mimoza

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 07 February 2013 - 08:44 AM

Don't you think it could be simply because your gene is very low expressed, if not at all?

#3 pcrman

pcrman

    Epigenetist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,165 posts
67
Excellent

Posted 07 February 2013 - 09:51 AM

How much RNA did you use for RT? You should consider increasing the starting RNA a bit. Using gene specific primers for RT reaction is another option.

#4 TV2013

TV2013

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 07 February 2013 - 11:00 AM

I have already thought on this but I would like to be sure that is only this. Do you understand?
Thanks

#5 TV2013

TV2013

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 07 February 2013 - 11:04 AM

Pcrman,

I have used 1ug of RNA for RT and my primer is specific for the gene.
Thank you




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

©1999-2013 Protocol Online, All rights reserved.