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qPCR on ChIP samples : problem with my Tm values!!


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#1 happipet

happipet

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Posted 03 October 2011 - 08:17 AM

Hi everyone,
I'm starting using this forum, and hope that you'll be able to help me...
I performed some ChIP experiments and found that my favorite transcription factor binds to the promoter of my gene of interest...
I tried to quantify this binding by qpcr analysis, but my problem is that i obtain some weird and highly variable Tm values...
I checked my primers efficiency by doing a standard curve with my diluted Input samples and all is ok (R2=96% , Eff=99%). The Tm values obtained with the standard curve are very closed to 90°C.
When I test my ChIP samples (each in triplicate), the Ct value for my Input sample is around 32 with a Tm comprised between 64°C and 89°C among my triplicates, and the Ct for my IP sample is around 39-40, with the same range of Tm values.. Ct values for IP IgG and no antibody samples are undetermined.
I can't interprete my results with such Tm values, because I can't know if it is due to non-specific amplifications or if it's the right amplicon...
If someone has already been confronted to such problems (and found a solution!!), please share your experience with me...
Thanks!




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