Hello everyone I have bisulphite converted my DNA using kit from Zymo research now I have to do methylation specific PCR. I have to check effect of my drug on methylation status of diff. genes compared to control.So, Do I need to quantiify the bisulphite DNA and then take equal amount for each sample for MSP or just use directly equal volume of it for PCR?
Please reply.
Thank you.
Dear Lalita,
There is no need to quantify bisulfite DNA if the starting amount of DNA used for conversion for all your reactions is the same. If your PCR conditions are standardized on bis-DNA you get after clean up from the above samples, then your PCRs will be consistent. Hope this helps.
Regards.
Pooja.
Dear Pooja,
Thank you for the reply. I am performing HRM and trying to get the % of methylation on various CpG sites. I too use Zymoresearch kit for BiS conversion.
However, when HRM is being performed the graphs are very inconsistent. What do you suggest might be going wrong?
Hi Lalita Sharma. You need to perform QMSP or Methylight PCR for quantify bisulfite DNA and one reference gene for normalization of DNA template in all samples. Hope this helps.
hi, you should quantify the elute after the conversion process using nano spectro or flurescence based method. this is spin column based and might not give you same yield every time you convert the DNA even if the starting material amount is same. best wishes.
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