I am having a problem with background drift with my Dual-labeled BHQ® probe. What might the cause be?
Background drift is natural phenomenon in qPCR reactions and does not indicate a problem with probe manufacture. Real-time instruments compensate for this drift through the process of baseline subtraction. Baseline subtraction usually involves extrapolating and subtracting a line rather than a constant value - a slope correction in effect. In some situations the issue relates to reducing agents like DTT carried over from the reverse transcription reaction since they are present in certain enzyme formulations like MMLV. DTT will degrade the Black Hole Quencher® (BHQ®) dye and cause the background signal to increase in a manner independent of amplification.