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
- - - - -

which exon to choose when designing primers for RT-PCR?


  • Please log in to reply
3 replies to this topic

#1 gyma

gyma

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 144 posts
2
Neutral

Posted 26 June 2011 - 05:50 AM

hi guys, I have a question when designing primers for RT-PCR. I want to check the endogenous level of one gene in different cell lines. To design the primers, I need to get forward primer in one exon and the reverse in another neighboring exon. But one gene may have several splicing isoforms and some isoforms may not have the exons that I choose. In this case, two sets of primers which have different target regions may give quite different results. Then how should I get a meaningful result?
another question is that gene information on splicing isoforms provided by NCBI and Genecard is different. Which one should I believe?
Thanks in advance.

#2 pcrman

pcrman

    Epigenetist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,165 posts
68
Excellent

Posted 08 July 2011 - 08:29 PM

Ideally, you should design your primers on the exons shared by most if not all isoforms or splicing variants. You can use UCSC genome browser which gives you better visualization of gene structures.

#3 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,201 posts
110
Excellent

Posted 11 July 2011 - 08:19 AM

Or Ensembl.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#4 gyma

gyma

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 144 posts
2
Neutral

Posted 14 July 2011 - 10:19 PM

thank you guys, I will try those tools.




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

©1999-2013 Protocol Online, All rights reserved.