When I over expression a protein and detect the increase of mRNA of that protein using real time PCR, the result show increase 1000+ fold to mock transfection sample. Is this result common and reasonable? I have over express several proteins, only particular proteins show too high increase in expression. When I was doing low cycle PCR and loading gel, I did not see the increase until that high (I have attach the gel picture). What can be the reason of that high increasing? I have using others pairs of primers also show similar high increasing.
Hi,
If you calculate relative fold change using the common delta CT method, and in the control group the gene of interest is absent ~ 40CT but in the treated group it becomes present - then you can get a 1000 fold difference BUT that change reflects from nothing to something.
What are the CT values for both groups?
Hi,
In my thought the gel data would be closer to what actually happened. Did you use the same primer set for both qPCR and regular end-point PCR? Then probably something's wrong with your qPCR reagents or procedures. Did you use a much more short amplicon for qPCR? Then I'd suspect contamination of your sample with the residual plasmid DNA, to which shorter amplicons tend to be more vulnerable. This can be easily tested by performing qPCR on RT minus control in parellel.
Cheers,
how did you work it 1000 folds ? is that how many cycle difference ?
How did you transfect , temporary or permanent will affect your result. if you use plasmid with tag you can look for the tag as indicator. if permanent you may have randomly inserted your transaction nearby promotor area. are you able to sequence or sort the cells ?
my ct value for protein A: control (21.64), protein A(12.36)
ct value of GAPDH : control (16.96), protein A (17.69)
i have use same primer for qPCR and regular PCR. The amplicon size is 154bp. The primer designed is located in two different exon (i think the mRNA/DNA contain the intron between this two exon can be eliminate by this primer set due to the long intron (>1000bp)). others samples without over expression protein A have quite similar ct value as control.
The transfection is temporary transfection, i transfected 1ug of protein A plasmid in 12well plate for 48 hour and then extract the RNA.
Hi, you mean you measured some kinds of RNAs by transient transfections. Under such the experiment, you should consider about the plasmid DNA contamination. Even if you treated the RNA with DNase I, the undesired amplification (i.e. amplification originated from the plasmid) will be occur at high possibility.
If you would like to test the expression levels of transfected plasmids, i strongly recommend to study protein (but RNA!) levels by Westernblot etc... if antibodies are available...
Good luck,
Mie
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