In RT-PCR the Ct value should be determined primarily by the abundance of the target cDNA rather than the concentration of your primers, which should be far in excess compared to the target cDNA. You could potentially increase the Ct value of non-specific primers by increasing the annealing temperature, but you won't be able to lower the Ct value of the specific primers.

Without knowing the details of your experiments, I don't think RT-PCR is the most straightforward approach to use for testing species identity (unless your samples contain a mixture of species). A regular PCR should work just fine, and the readouts will be presence/absence of a band from the species-specific primers. The proper way to ensure specificity would be to increase the annealing temperature, but you can also reduce non-specific signal intensity by reducing the number of PCR cycles.

Have you tried simply diluting your cDNA samples more ? If you have too much DNA to begin with, you may get very low Ct values but bad curves. Dilution would higher the Ct and may give better curves.

HI,

I agree with the above views. It is also not also clear if you are using cDNA or DNA. For species identification using certain loci, I will assume you are relying on genomic DNA for the analysis. Here, copy number of the gene/region of interest will also decide the Ct values. For example, if rDNA spacer regions are used (copy number: 100s to 1000s), dilution will be needed. So see the copy number of your gene of interest, optimize, include the suggestions listed by above researchers and then finalize the protocol

good luck