qRT-PCR Problems - (May/07/2009 )
Hi, I have been doing ChIP and mRNA assays with qRT-PCR. I am new to this and I have having alot of problems. I have horrible melting curves and the differences between repeats are quite large. What do you guys think is the problem?
this is not easy to answer...at least for me indeed ,your melting curves look very different from mine. but I only do quantitative real-time pcr and i know nothing about chip.
if this picture is from anmelting curve of rt-qpcr ,then it really looks strange.
i would check the sample without template and look at the melting curves. if there is a peak, this would mean that your primer form dimers ,which is bad (simply spoken).
normaly you would anticipate one peak in the samples with cdna in it ,namely the meltingpoint of the amplicon taht you amplified.
it could of course also be that your cDNA isnot good.
bad templates would mess up the whole reaction of course