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To isolate DNA, RNA and Protein from same culture flask for methylation study?


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#1 genie

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Posted 05 August 2011 - 08:23 PM

Hi All,
I am going to study the methylation of the promoter of a gene of interest using a cancer cell line. I want to correlate the methylation pattern of the promoter with the protein expression and the mRNA expression. To do this do I have to ensure that I isolate the DNA, RNA and Protein from cells grown in the same culture flask. Or can I isolate them separately from different flasks for more yield.
Is there some rule to this?

Can passaging have an effect on methylation, like aging? If any one is working on similar lines, please give me a reply. PS: my cells are growing really fast, so please reply soon. Thank you guys a million :)

#2 methylnick

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Posted 07 August 2011 - 06:37 PM

the ideal experiment would be to extract DNA, RNA and protein from the same cells you are studying, trizol would be the way but not optimal,

one way we get around this is to divide you culture into three from which you take one for DNA the other for RNA and the final one for protein.

Passaging does have an effect on DNA Methylation potentially. there are a number of papers in the literature about this.

nick

#3 genie

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Posted 07 August 2011 - 07:08 PM

the ideal experiment would be to extract DNA, RNA and protein from the same cells you are studying, trizol would be the way but not optimal,

one way we get around this is to divide you culture into three from which you take one for DNA the other for RNA and the final one for protein.

Passaging does have an effect on DNA Methylation potentially. there are a number of papers in the literature about this.

nick


Hi Nick,
Thanks for your suggestion. and I did come across this paper,"DNA Methylation Decreases in Aging but Not in Immortal Cells", WILSON and JONES, 1983,
When normal diploidfibroblastsfrom mice, hamsters, and humans were
grown in culture, the S-,nethylcytosine content of their DNA's markedly decreased.
The greatest rate of loss of 5-methylcytosine residues was observed in mouse cells,
which survived the least number of divisions. Immortal mouse cell lines had more
stable rates of methylation.
That paper put my fears to rest. -_-




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