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Picking primers to confirm Illumina meth27 results


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#1 white048

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Posted 30 September 2010 - 12:40 PM

Hello everyone,

First a disclosure - I am an MD not a PhD and have a very simple understanding of the technical aspects of this field. Please accept my apologies in advance.

I have some CpG sites on some genes that were differentially methylated between cases and controls as run on my Illumina Meth27 platform. I now need to replicate these findings ( just a few of these not all statistically signif ones) with either MSP or pyrosequencing. Do I use the sequence from Illumina that correpsonds to the CpG that was differentially methylated and design primers based on that?

Is there a place I can go to see if others have already designed these primers and have a specific PCR protocol that already is know to work? Or do I have to troubleshoot each of these reactions myself?

Thanks Wendy

#2 methylnick

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Posted 30 September 2010 - 01:23 PM

Hi Wendy. We have over 200 assays validating infinium methylation. Pryo would be better than msp as it is more quantitative. There is no website. I can share with you primers that we use.

We design up and downstream of the probe location by 500bp. We generated from the manefest file a bed table for ucsc.

Would love to chat to you more about what you are doing using infinium and plans for the 486k array

#3 white048

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Posted 04 October 2010 - 07:32 AM

Hi Wendy. We have over 200 assays validating infinium methylation. Pryo would be better than msp as it is more quantitative. There is no website. I can share with you primers that we use.

We design up and downstream of the probe location by 500bp. We generated from the manefest file a bed table for ucsc.

Would love to chat to you more about what you are doing using infinium and plans for the 486k array



Thank you! Let me figure out what genes I want to replicate and let you know. So it sounds like you do amplify around that same site. I am not sure what this sentence means - "We generated from the manefest file a bed table for ucsc."

As for the 486k chip, we just wrote an R01 to use this chip - I'm keeping my fingers crossed! Is it allowed to talk directly email to email? Not sure about the etiquette of such things and do not want to offend or bother you. Wendy




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