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Does aza affect DNA/RNA quality? - (May/07/2007 )

Dear all,

as the question inthe title asks: does aza treatment of cells affect the quality of the DNA and RNA? It certainly affects the yield (higher aza conc. lower yield of DNA and RNA because of cell numbers) but does it affect the quality as well for downstream applications?

Whenever I do a bis. conversion of aza treated DNA, the PCR bands on my gel afterwards are always of less intensity (and more smear) than my untreated samples eventhough I used the same quantity of DNA for the conversion. The phenomenon continues in the quality of the number of sequencable plasmids and the sequencing results afterwards.

Can anybody offer an explanation?

-Bram-

Does this have anything to do with some researchers giving the cells an extra day with normal media to let the cells recover?

-Bram-

Hi Bram,

I have seen a reduction in the number of cells and amount of DNA and RNA in my experiements and I have put this down to most of the cells dying from the drug. I haven't seen the effect passed onto sequencing so I don't think azaC would affect the sequecing, because the PCR stage would effectively wipe out any azaC incorporated into the DNA. I am not too sure if azaC reacts with bisulfite, it's possible.

and the recovery after azaC treatment is precisely what is done so that the cells that are still living just, can grow a bit, with our cells, we know they divide once a day (approximately) so this is fine. You don't want the cells to divde anymore because the genes you are looking to demethylate would most likely remethylate in that time.

Nick,

-methylnick-

Thanks Nick

-Bram-

QUOTE (Bram @ May 10 2007, 12:26 AM)
Thanks Nick


Hi Bram,

I am suffering from the same problem as you, and was wondering if you've found a solution to it. I am really puzzled by this. Wonder if it's the operator's technique or something.

Thanks

-phaffia-

Hi Phaffia,

I've basically changed my technique for methylation analysis. I'm now using pyrosequencing instead of cloning and I haven't encountered any major problems so far (knock on wood) with AZA treated cell DNA. I'm still not sure what the cause of the problem was to be honest.

Sorry I can't be of more help,

Bram

-Bram-

QUOTE (Bram @ Dec 14 2007, 03:04 AM)
Hi Phaffia,

I've basically changed my technique for methylation analysis. I'm now using pyrosequencing instead of cloning and I haven't encountered any major problems so far (knock on wood) with AZA treated cell DNA. I'm still not sure what the cause of the problem was to be honest.

Sorry I can't be of more help,

Bram


No prob Bram. Thanks and all the best!

Phaf

-phaffia-