5 replies to this topic

[Louie]

Hi

I really need to find a promoter region for a human housekeeping gene that would be suitable to use as an unmethylated control in a MeDIP. I have been looking through commonly used genes such as beta-actin and GAPDH but they seem to have methylated promoter regions. Not sure what is normally done in relation to this, do people just desing primers to avoid the methylated areas? I am finding this hard at the moment so any help would be greatly appreciated.

Thanks

[methylnick]

why not an imprinted region where you expect equal enrichment of the seuqence between the non-bound to bound fractions

[SarahWaite]

Louie, on Oct 27 2009, 10:33 PM, said:

Hi

I really need to find a promoter region for a human housekeeping gene that would be suitable to use as an unmethylated control in a MeDIP. I have been looking through commonly used genes such as beta-actin and GAPDH but they seem to have methylated promoter regions. Not sure what is normally done in relation to this, do people just desing primers to avoid the methylated areas? I am finding this hard at the moment so any help would be greatly appreciated.

Thanks

Hi, Do you have a reference showing that GAPDH promoter is methylated? I am looking at methylation and I need to know if GAPDH is methylated or not.

Many Thanks

Sarah

[ForEpi]

I can't give you a literature reference (currently writing up the paper) but I have analysed GAPDH and ACTB in prostate cells and they are indeed methylated. For our MeDIP validation experiments we used SELS (Selenoprotein S; pyrosequencing) which is not methylated. Relative to SELS, we obtained a 50 fold enrichment of GSTP1 methylated fragments (DNA QPCR). Be aware though that SELS is active in the inflammatory pathway and so increases expression after 5-aza or dAZA treatment. If you need primer sequences let me know.

Hope this helps.

[racingstud]

methylnick, on Oct 29 2009, 10:51 AM, said:

why not an imprinted region where you expect equal enrichment of the seuqence between the non-bound to bound fractions

Could you also just use a region of DNA where there are zero CpGs or is there to high of a risk that non-CpG methylation could be present.

[SarahWaite]

ForEpi, on Nov 4 2009, 07:14 AM, said:

I can't give you a literature reference (currently writing up the paper) but I have analysed GAPDH and ACTB in prostate cells and they are indeed methylated. For our MeDIP validation experiments we used SELS (Selenoprotein S; pyrosequencing) which is not methylated. Relative to SELS, we obtained a 50 fold enrichment of GSTP1 methylated fragments (DNA QPCR). Be aware though that SELS is active in the inflammatory pathway and so increases expression after 5-aza or dAZA treatment. If you need primer sequences let me know.

Hope this helps.

Thanks this is really helpful, could you let me know the primer sequences and also where you have located your primers - I am currently working with canine DNA and G3PDH is the canine homologue to GAPDH and so hopefully I can use the methylation pattern / location of GAPDH to infer were the methylation is in G3PDH.

Many Thanks

Sarah