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How to determine the result of my msp? - methylated or unmethylated? (Nov/08/2005 )

Hi
I am doing a tumor suppressor gene methylation research.
Bisulfite modificated DNA from cancer tissue was amplified with MSP primer.
When the PCR cycle is 35~36,many cancer samples were double bands with bright methylated and unmethylated bands.Sometime unmethylated band was more bright and bigger than methylated band.
When I reduced the cycles to 32~33,there was very faint bands,even some can't be visualized in 2% gel.
So I have to increase the PCR cycle (35).now how to explain my MSP result? Can I decide that all the samples with bright two bands are methylated??????
In my opinion ,because of the contamination of normal cell in cancer tissue,most of the MSP result are supposed to be two bands.am I right????

-rockysofar-

yeah that's unfortunate.

if you can get a pure population of tumor cells you will get the black and whiteness of methylation and no methylation.

The results you are getting are not conclusive though I have to say, maybe the primers are suboptimal in the given reaction conditions, you could try and optimise this, Tm, Mg conc etc...

N

-methylnick-

yeah

When I amplified the template from tumor cell line with the same primer and identical PCR reaction condition.I truely got the black and whiteness of methylation and no methylation.
That is to say my PCR condition is optimal???

-rockysofar-

Isn't it also possible that the bands you see in both methylated and unmethylated refer to the fact that maybe your DNA is methylated for an allele and unmethylated for the other?

-Nexuuus-

absolutely,

it can also say you have a mixed population of cells of which some are methylated and some are unmethylated at that particular locus.

-methylnick-

QUOTE (methylnick @ Dec 3 2005, 08:05 AM)
absolutely,

it can also say you have a mixed population of cells of which some are methylated and some are unmethylated at that particular locus.


You probably need to sequene the PCR product to see whether it is allele specific methylation or partial methylation. Have you done 5-aza induction?

-akahst-