Hi everybody. I'm having some big problems with my qPCR and I need some help please.
I'm trying to amplify and quantify a HOX gene, whereas the positive control (colon) amplifies early in the Ct22 and H2O doesn't amplifies anything.
The problem I'm dealing with is "late amplification" of my cDNA that starts at Ct 35-36. My endogenous control (GUS) amplifies at Ct25. So I would discart these results for its late amplification.
The matter is when I run a gel, a band appears at the same size of C+. And so in the negative tissue control. No band appreciated in the H20 lane.
I'm using a comercial assay (probe+primers) in a 7900HT thermocycler applying standard PCR conditions in a 20ul volume per well. I've reapeated the previous RT-PCR twice separating my samples from the C+ in order to avoid cross contamination.
I have not so much experience in this field and I would thank some help!
Thank you all!