Hi to everyone,
I'm recently investigating some genes that control Shh activity using RT-PCR technique.
I normalised my genes with reference gene GAPDH.
My values are good, cp values all from 19 to 26, with low variations in triplicates and good melt curves but negative control of GAPDH is around 21-22-23... Is that mean I have amplification even if I used RNASe free water as -ve control?? If I look at the melt curves, I have a threshold but the curves are very low compared to those of the other samples, and only in few of -ve control Gapdh curves I have a proper melt curve.
I also did another run in which one sample for sure it's contaminated ( I don't have cp values for my genes or the cp values are around 33-37) but if I look at Gapdh, I have values around 21-23 and -ve still around 21-22-22.
I really can't find an answer to explain that. Could you help me to understand that?
You are seeing amplification of your no template control using the GAPDH primers? Then you have contamination. Throw away ALL of your reagents (water, primers, buffer, etc.), clean your work station, don't use your qPCR pipettors to load gels, and try again. Good luck!
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