Why do I get different Ct values for the same probe sequence when labelled with a different dye?

It is not unusual to observe slightly different cycles to threshold (Ct or Cq) values for the same probe sequence labelled with different fluorophores. Such variation is typically on the order of 1 to 2 cycles and relates to the differences in dye intensity as well as the variation in instrument optics across the different channels. Fundamentally, all real-time thermal cyclers are engineered to detect fluorescein (FAM) first and foremost, so dyes with longer wavelength emission may be detected less sensitively. Occasionally, changing the fluorophore can have a profound impact on functional performance, particularly when the melting temperature of the probe is marginal. Such an outcome might relate to the hydrophobic attraction between modifications, or a change in melting temperature with the new fluorophore.