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5AZAdC treatments - (Oct/21/2006 )

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Hi, I'm really new with methylation work and is really excited to see the discussions going on here!

I read a few topics on 5AZAdC treatments and it seems like it's a norm to be treating the cells with fresh 5ZA daily as they have a short half life. Do you guys check for cytotoxicity?
I read some papers where their protocols were to treat for 24 hours , and therafter, replacing with drug-free media, subcultured at equal densities at different number of daysafter initial treatment.

May I know the purpose of maintaining the cells in drug-free media? I'm a bit worried as I do not do that for my experiments!

Thanks very much

-sharonpek-

hi sharonpek, welcome to the forum!

I certainly check for cytoxicity by titrating 5azaC over three days and measuring cell death. as for recovery, 5azaC is quite a nasty drug that affects many things other than demethylation. so I have seen assays whereby intial demethylation from 5azadc persists and the cells are able to recover with the genes reactivated.

N

-methylnick-

QUOTE (methylnick @ Oct 22 2006, 10:05 PM)
hi sharonpek, welcome to the forum!

I certainly check for cytoxicity by titrating 5azaC over three days and measuring cell death. as for recovery, 5azaC is quite a nasty drug that affects many things other than demethylation. so I have seen assays whereby intial demethylation from 5azadc persists and the cells are able to recover with the genes reactivated.

N


thanks methylnick
Is there any particular assays which u perform for cytotoxicity, or do u just do a trypan blue counting?

And, may I know what is the maximum number of days which u had treated your cells (daily) for?
I realised my cells re-expresses the genes between 1-3 days. Further treatments for 4-5 days didnt not re-express the gene further. In fact, it seemed to be down-regualted !! happy.gif
Is that a norm?

Thanks very much

-sharonpek-

Hi Sharonpek,

my titrations ran over three days and I performed trypan blue staining and counting to determine viability.

I think what you are seeing after 4-5 days is saturation, whereby the promoter of your gene of interest can only be demethylated such that it is unmethylated and hence reach maximal reactivation by 5-azaC, you could try adding a HDAC inhibitor as well such as TSA that usually synergistically reactivates the gene than just 5-azaC alone.

Nick

-methylnick-

thanks Nick! TSA would definitely be in the next part of my experiment smile.gif

-sharonpek-

Hi, One more question.
I've got a significant re-expression of my gene at 5uM after 2 days of treatment but the viability is pretty low.
My gene of interest might be inducing apoptosis so how can I determine if the the low viability is due to cytotoxity or cell death due to the gene re-expression, without doing any sophisticated tests like TUNEL?
I'm just wondering as I'm not exactly sure if 2 days of treatment without "recovery days" is good enough?

Thanks

-sharonpek-

the only way to find out is to do the experiments,

it is possible that the high cytotoxicity is effecting your expression, also it's highly probable that the RNA obtained after aza treatment could be of low quality and this could be reflected in the Ct values you are getting.

you could try harvesting the cells after 1 day of treatment or indeed allow for "recovery" but as these are cell-line and gene specific, you would have to optimise the conditions to taylor your experimental system.

Good luck

Nick

-methylnick-

Hi, I've obtain some realtime PCR data from RNA extracted from 5AZA-treated lines, with dosages 1,5, 7 and 10uM. There's a modest re-expression as compared to DMSO control (about 2 folds increase). I've read papers that have robust re-expressions of thousand folds! We've examined the promoter region of my gene of interest; most of the CpG islands are methylated. Will go on to look at the DNA from the treated lines.

I would like to know if "modest" re-expressions are common/interesting as we are not exactly sure if methylation is indeed an important event in controlling the expression of this gene. Thanks smile.gif

-sharonpek-

I think modest re-expression would depend on the basal transcription level.

I think the papers you are referring to are in fact looking at that gene being off, and then switched on by 5-aza-dC give such a jump,

if you getting 2-fold and it is a consistent result then i would say it is real albeit modest.

N

-methylnick-

Hi! I am also new in epigenetic study.

As I check some documents to describe about azadC treatment, it said that azadC need to prepare freshly before each time use. Is there anyone can give some experimence about the preparation and reservation of azadC stock?

Another question is that after I treated a common used breast cancer cell line MDA-MB-231 with azadC for 5 days and refresh drug daily, I still can not remove methyl group from CpG island in the Estrogen Receptor gene promoter effectively after MSP detection as other paper shown previously. wacko.gif Could anyone give me some suggestion?

Thanks a lot

George

-georgewu-

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