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methylating agent


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5 replies to this topic

#1 SF_HK

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Posted 09 July 2009 - 07:34 PM

Hi,

My gene of interest shows reduced expression in my normal cell line but overexpression in my cancer cell lines. have heard of de-methylating agents. Are there any methylating agents out there? I would like to test if teh overexpression of my gene is due to demethylation? How can I approach this?

Thanks

#2 Rsm

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Posted 09 July 2009 - 08:48 PM

Hi,

My gene of interest shows reduced expression in my normal cell line but overexpression in my cancer cell lines. have heard of de-methylating agents. Are there any methylating agents out there? I would like to test if teh overexpression of my gene is due to demethylation? How can I approach this?

Thanks

Well, first I would suggest you to check the methylation status of your promoter of interest in both normal and cancer cells. There is a lot of possibilities for overexpression of a gene, epigenetics is just one of them. And before you waste your time and money, better check first... Bisulfite sequencing seems to be quick and rather inexpensive. You can also check many CpG islands at once.
I never heard of methylating agents... The only ones I know of are DNMT1 and 3 :wacko:

Cheers,
Minna
I got soul, but I'm not a soldier

#3 SF_HK

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Posted 10 July 2009 - 12:59 AM

Hi,

My gene of interest shows reduced expression in my normal cell line but overexpression in my cancer cell lines. have heard of de-methylating agents. Are there any methylating agents out there? I would like to test if teh overexpression of my gene is due to demethylation? How can I approach this?

Thanks

Well, first I would suggest you to check the methylation status of your promoter of interest in both normal and cancer cells. There is a lot of possibilities for overexpression of a gene, epigenetics is just one of them. And before you waste your time and money, better check first... Bisulfite sequencing seems to be quick and rather inexpensive. You can also check many CpG islands at once.
I never heard of methylating agents... The only ones I know of are DNMT1 and 3 :wacko:

Cheers,
Minna


Hi,
Thank you for your response. I agree with you that there could be several reasons for overexpression. What is the accepted way to check teh methylation status of the promoter? Is it treatment with 5'azacitidine? or bisulphite sequencing? If I use 5'azacitidine, I should expect expression in cell lines don't have reduced expression and no changes in expression in cell lines that show overexpression?

Sorry if any comments appear silly. I'm new to this area of research.

#4 Rsm

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Posted 10 July 2009 - 08:27 AM

5-azacytidine and bisulfite sequencing are two completely different things, as you might know. To check the methylation status of your promoter, you usually use bisulfite sequencing (or other methods, like methylation-chip or this restriction enzyme, the name I just forgot...). If you find different methylation levels, you can try treatment with 5-aza (DNMT inhibitor) and see if that changes the expression of your gene. But better start with bisulfite.
Cheers,
Minna
I got soul, but I'm not a soldier

#5 SF_HK

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Posted 11 July 2009 - 07:51 PM

5-azacytidine and bisulfite sequencing are two completely different things, as you might know. To check the methylation status of your promoter, you usually use bisulfite sequencing (or other methods, like methylation-chip or this restriction enzyme, the name I just forgot...). If you find different methylation levels, you can try treatment with 5-aza (DNMT inhibitor) and see if that changes the expression of your gene. But better start with bisulfite.
Cheers,
Minna


5-azacytidine is a demethylating agent and thus wouldn't it better to do a quick experiment by treating my cell lines with 5-azacytidine to check is they are methylated or not? I mean there would be a change in expression if they are methylated and no change in expression if they are not methylated? Isn't this faster than bisulphite treatment? And then this experiment could be followed by bisulfite sequencing?

#6 Rsm

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Posted 12 July 2009 - 02:52 AM

Well, 5-aza is a Dnmt inhibitor, and does not induce demethylation per se. But that's nitpickery...
Sure you can treat your cells with 5-aza and check if there's a change in expression. I guess that is really quicker than doing bisulphite. If there is a change in expression if a promoter is methylated or not, is actually under scientific discussion. Most people agree that methylation inhibits gene expression, but there seem to be rare cases where methylation induces expression of certain genes.
I would start with bisulphite, but you seem to be very dedicated to do 5-aza, so go for it! But make sure you get reproducible data... :P
Best,
Minna
I got soul, but I'm not a soldier




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