Hello, I am experiencing a weird problem with strand-specific RT-qPCR. I keep getting a lot of signal in the "no-RT control" for the gene of interest (HIS3) while I am getting nothing in the "no-RT control" for the house-keeping gene (ACT1).
The RT step (after Turbo DNase digestion of total RNA extract) for both genes was done in one tube and the cDNA of that tube was used for the qPCR. There should be no signal in the "no-RT control" for both genes but this is not what I got. I got a signal in one and not in the other as I mentioned. This was consistent with 6 biological replicates. I repeated the whole experiment with newly ordered primers to rule out the contamination of the primers with gDNA and I got the same thing again. The no template controls are fine with no signals. The organism is S. cerevisiae.
Any ideas why this could be happening?
Thanks in advance.
Thanks Matthias and Jiang,
I meant SYBR signal from the qPCR.
The melting curve for the two is exactly the same.
The signal in the no-RT is weaker than the RT (difference is 5-6 Cq) but is still too high for a no-RT. And it is increasing while cycling like a normal curve.
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