I am using Sybr Green for my qPCR. I already have the optimal conditions for the assays (T and primer concentration). I am using the amount of cDNA corresponding to 150ng of total RNA because the gene of interest has very low expression.
The negative control (NTC) is Ok. However I have fluorescence signal in RT control, similar to the signal for cDNA. We thought it was genomic DNA and we used 2 methods to fix the problem. We used DNAse and also some kit from Qiagen to eliminate gDNA. After the treatments the problem is the same.
If I dont have cDNA, or gDNA in my RT control, then, what is the template for the polimerase?? The RNA?? Maybe I have to use RNAse after cDNA synthesis.
I would like to know If somebody has some suggestion. I am thinking that there is some kind of science fiction in my tubes...It is not contamination because my NTC are clean..
RT control positive!