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Confirm DNA enrichment of methylation array


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#1 bactin2

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Posted 06 March 2009 - 01:09 AM

Hi all,
I am doing the methylation array(agilent platform).here we pull down the methylated DNA by IP.but before moving on to adding the dna to the slides which are very expensive as we all know,i wish to confirm if the methylated DNA are enriched,so how do i confirm it.IS MSP possible,if i know any one of genes to be methylated through other publication,can i look for the methylation status of that gene using MSP to confirm that all is going well before adding the IP dna to the array slides.

Or should i do a real time PCR for a specific gene promorter region

thanks.

#2 pcrman

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Posted 15 March 2009 - 10:00 AM

What do you use to pull down the DNA, 5mC antibody or else? I guess what you pulled down is methylated DNA, right? Then you just need to amplify some known methylated promoters. I don't know what method you used to pull down the DNA and whether the DNA has been modified or not. If the DNA has not been bisulfite modified, you can use regular ChIP primers to amplify potentially methyated DNA sequences. If the DNA has been bisulfite modified, then you will need to use BSP primers to do the PCR.

#3 march

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Posted 23 March 2009 - 05:31 PM

What do you use to pull down the DNA, 5mC antibody or else? I guess what you pulled down is methylated DNA, right? Then you just need to amplify some known methylated promoters. I don't know what method you used to pull down the DNA and whether the DNA has been modified or not. If the DNA has not been bisulfite modified, you can use regular ChIP primers to amplify potentially methyated DNA sequences. If the DNA has been bisulfite modified, then you will need to use BSP primers to do the PCR.


I am using the 5-methylcytosine antibody to pull down.With regard to the primers,i should design from the promorter region of a known methylated gene.As for the control,can GAPDH be used as an internal loading control and the PCR results should be in such a way that the known methylated gene expression is more in the IP sample than the Input sample but will GAP appear APPEAR EQUAL IN BOTH THE IP DNA SAMPLE AND THE INPUT DNA SAMPLE.




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