designing a methylation study - (Aug/13/2013 )
hello to all,
I am planning a methylation study but very new to the subject. I will do gene specific methylation study. I used methprimer (http://www.urogene.org/methprimer/) , I took the first 4000bp region of ABCA1 gene and methprimer gave me 7 CpG islands in the region. Enclosed, you can find the region I put in methprimer.
Is it OK to if I take only promoter region for a methylation study or do I have to look for CpG islands in whole gene?
Do I have to build primers for all CpG islands seperately?
The primer suggestions in methprimer spans only 1st and 2nd islands,why is it so?
You don't need to examine such a large region of the promoter. If you use a 4000 bp input sequence, the program just returns primers which sore highest unless you specify where the target sequence will be. My suggestion is to focus on the region surrounding the TSS, especially CpG-rich areas. Even if you want to look into other regions of the promoter, you can design primers piece by piece.
It is generally assumed that methylation near TSS has an greater effect on transcription than that of distal areas. But a recent genomewide study seems to suggest otherwise. Please see this recent paper: Charting a dynamic DNA methylation landscape of the human genome. If what observed in the paper is true, then it is worthwhile mapping distal CpG islands such as those near enhancers.
Pcrman, thanks a lot for the prompt reply.
I read the article. The gene is huge (141,425 bp), it was tough to search the whole gene for CpG islands. I used ensembl to visulaise the exonic regions. The CpG islands mostly gathered in promoter and first exon, there is one near the 2nd exon. I ended up wit 9 CpG islands. How should I proceed from here, do you think I should design primers for all and then proceed with bisulfite sequencing, I would be grateful if you share your suggestions
You can start with the CpG island which is in the promoter and closest to TSS, although other area surrounding the TSS including the first exons/introns could also be important. I mentioned the paper just to let you know that there is new information which contradicts existing knowledge about the effect of methylation and gene transcription.
OK, thank you for the suggestions :)
I have measured ABCA1 core promoter previously. You can get the primers that worked and the cycling temp at:
Tobi EW et al. Human Molecular Genetics 2009