eGFP transcript quantification using LightCycler RT-PCR

Fernando king-ferdi at gmx.net
Tue Feb 7 06:53:16 EST 2006


Thanks for the fast answer,

At the moment I am using one of my RT-PCR primers for generation of the 
cDNA. So I guess I will try another - further downstream primer - which 
includes the whole PCR product in the first step.

I have two positive controls: The first one expresses eGFP at quite a 
high level, the second one expresses eGFP at quite a low level. I've 
problems with unspecific bands mainly in the latter one, even though the 
specific product appears earlier than the unspecific one. So I am able 
to quantify the positive controls, but the background is too high for 
the actual experiment.

My negative controls are one containing H20 instead of template and one 
containing untransfected CHO or HeLa respectively. The noise only 
appears in the latter ones. Since the RNA isolation includes a DNAse I 
step I'm quite sure that there's no problem with genomic DNA carry over.

Fernando



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