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.