As a preliminary experiment for qRT-PCR, I performed qPCR using synthetically ordered amplicons for GAPDH and HER2 as templates. Aside from differences in template and primer sequences, all reaction conditions were identical, with a final template concentration of 1 pM and a final primer concentration of 200 nM.

While the Ct values are nearly identical, which i suppose that there is no difference in PVR amplification efficiency. I observed differences in fluorescence intensity between the two targets. Notably, the GAPDH amplicon(103nt) is about 20 nucleotides longer than the HER2 amplicon(84nt).

Since I plan to use the ΔΔCt method, I’m wondering whether this variation in fluorescence intensity could affect my results. Could this difference impact Ct value determination or compromise the reliability of the ΔΔCt analysis? What factors should I consider, and how can I ensure accurate interpretation?

Thank you in advance for your insights!