What factors affect Cy3/Cy5 relation?
You should not expect a constant Cy3/Cy5 ratio over a wide signal range. The Cy3 and Cy5 dyes have different molar extinction coefficients and quantum efficiencies. Fluorescence intensities of the concentration of spots from each tend to produce a sigmoidal shaped curve. However, the sigmoidal shape is not identical for each dye and is more importantly influenced by environmental factors as – pH, oxygen content, temperature, buffer strength, quenching, solvatization, etc. If any of these variables change, the intensity of fluorescence changes differently for the two complexes. Therefore it is essential to keep the same conditions for each experiment. Additionally the reactivity from boths dyes to DNA is differently. The AT content has been shown to affect uptake rate differently between Cy3 and Cy5. Moreover, some scanners (e.g. Tecan LS400) have different laser powers for the red and green channel.
