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I am having over-expression while expecting silencing in real time qRT-PCR... wh

qRT-PCR silencing real time over-expression

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#1 kutayozturk

kutayozturk

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Posted 11 June 2012 - 09:32 AM

Some time ago I have run qRT-PCR to determine silencing level of my gene of interest, then I observed over-expression in all of my samples. After reading a bit, I realized that the problem might be caused by insufficient DNase treatment. I have changed my DNase, and confirmed that it works efficiently by doing PCR on 1 uL of DNase-treated RNA, seeing no bands. I have also ordered 4 more normalization primers, and checked their expression level between control and silenced groups by using GeNorm, then I have chosen the most stable two. (around M=0,5) Today I have repeated the experiment again, and I have seen over-expression in some of the samples. Even if there is no silencing, I must see the same expression level between control and silenced groups. Over-expression means something is absolutely wrong! I don't have too much cDNA to test, I can only run qRT-PCR a few times more. This is why I have to make sure everything before doing another qRT-PCR. Can you please help me? What could be the reason, even if the silliest one? I am seeing good dissociation curves, but i can still try another primer set (if you think it is worth to spend cDNA on it). I will run the qRT-PCR products on gel anyway, to make sure if I have one product or not. If you need any more information about the experimental details, I will be glad to tell.





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