qPCR inhibition - (Mar/19/2011 )
Im having trouble with my qPCR assays. It seems like I'm getting a inhibitor in some of my samples when I'm producing cDNA. I use Invitrogen kits to DNase treat my samples then produce cDNA and sometimes after this the samples are fine, but other times the standard curves I make from these sample are completely off. The first few dilutions won't amplify properly and only by the 4th or 5th dilution do I get a normal sigmoidal curve. Its not due to inhibition in the original sample because I have produced cDNA from the same RNA sample and once its been fine, but the second time it wasn't. I can't work out what is causing this because I'm only adding reagents from the invitrogen kits. Does anyone have any idea of what could be going wrong?
what about the concentration? ... maybe it is highly underestimated?