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
- - - - -

Problems with the study of promoters methylation


  • Please log in to reply
1 reply to this topic

#1 Robert

Robert

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 08 January 2016 - 02:43 AM

I would like to examine whether the promoters of isoform A and B of the progesterone receptor are subject to methylation in the corpus luteum during the estrous cycle in cows. I designed the primers for each isoform to methyl specific PCR. Methylated and unmethylated specific primers were designed with using MethPrimer web page. I performed PCR using Hot Start Page polymerase and bisulfite converted genomic DNA as template. The obtained results were demonstrated partial methylation of both isoforms. Both pair of the primers for methylated and unmethylated sequence showed bands on agarose gel. The assessment of the degree of brightness of the bands on the agarose gel were suggested not 50% methylation. I wanted to confirm these results by sequencing the fragments of the promoters of both isoforms of DNA after conversion and compare to the reference DNA to see what percentage of DNA is methylated in both promoters of A and B isoforms. However, sequencing revealed that the both promoters are not methylated (100 % unmethylated). Is this situation is at all possible. I'm trying to find a solution and nothing comes to mind.



#2 methylnick

methylnick

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 266 posts
6
Neutral

Posted 12 March 2016 - 07:20 PM

Hi Robert, sorry for the hiatus. 

 

Do you have known controls to ensure your primers sets actually work? It is possible to have heterogeneous methylation. 

 

Nick


All comments and communication are my own personal ones, and are not tied to any of my affiliations. 





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

©1999-2013 Protocol Online, All rights reserved.