Which factors influence relative probe signals?
The relative probe signal is the peak area of a probe amplification product divided by the peak area of all probe amplification products in that sample. Probe design is a major factor influencing relative peak signals (Schouten, J.P. et al; NAR 30, e57). In addition, the quality of the probe oligonucleotides also has a strong effect. Long (>130) synthetic MLPA probes usually result in low peaks. Therefore, the SALSA MLPA probes made by MRC-Holland consist of one synthetic, relatively short fragment, and one longer, M13-derived fragment. These M13-derived long oligonucleotides are of much better quality than synthetic counterparts. Thirdly, the amount of KCl and Polymerase present during the PCR reaction can also influence relative probe signals. In addition, there is also a relationship between relative probe signals and polymerase activity. The relative signal strength of some probes may decrease with reduced polymerase activity; whereas other probes may actually have higher signals.
