I am attempting to validate existence of a transcript using 40 cycle qPCR. I designed primers for a unique feature of this transcript, and also designed primers for a sequence in the transcript that is common to several transcripts of this gene, and finally I used GAPDH as a control. I already knew beforehand that this transcript has very low expression.
For the unique region, my ct values for primer 1 were between 31-34.8, and my ct values for primer2 and primer 3 were all consistently 35, and primers 4-6 gave no result at all.
For the region that is common to several transcripts of this gene, the ct values were between 30-34 for one primer, and consistently 35 for another primer.
For GAPDH, ct values were around 22.
Can I use these results to indicate that my transcript exists (or even may exist?) in the samples I tested? Is the fact that the ct values for the region unique to the transcript I am targeting and the region in common between several transcripts are quite close indicative of anything? On the graph from the qPCR, there are distinct rises respectively around 33 and 35 for the unique region amplicon.
EDIT: I was doing a bit more research, and it appeared that this study: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4296690/
also had results that came up "late cp call last five cycles has higher uncertainty" and considered these results positive if they could be replicated in a duplicate - if I were to show this as well, can I use this data in a paper?