Variability can be introduced by a number of factors, including sample treatment, the presence of impurities in the reaction, experimental issues, and probe(mix) characteristics.
Relative probe signals are constant as long as the reaction conditions are the same. Differences in reaction conditions do not always affect all probes equally, which can lead to changes in relative probe signals. For example, there is a relationship between relative probe signals and polymerase activity. The relative signal of some probes may decrease with reduced polymerase activity, whereas other probes may actually produce higher signals. A relatively common source of variability comes from the presence of impurities in the sample DNA. Impurities, such as salts, ethanol, EDTA, and phenol/trizol, can affect one or more steps in the MLPA reaction. Examples include a reduction in polymerase activity, altered denaturation efficiency or a reduced ligase activity. Reducing the amount of DNA (to 50 ng minimum) or performing an extra DNA clean-up step may have a beneficial effect on the variability.
Differences in the treatment of samples can also introduce variability. DNA extracted from a different tissue source or with a different extraction method may have different properties or impurities. Therefore, it is important to always treat all samples in an experiment as similarly as possible. This is especially critical for DNA extracted from formalin-fixed, paraffin embedded (FFPE) tissues, as this DNA is often relatively impure and as it may have been chemically modified.
Variability can also be the result of experimental issues such as pipetting errors, improper mixing or equipment malfunction. Therefore, it is important to prepare master mixes whenever possible, mix everything properly, and to ensure that calibrated pipettes and thermocyclers are used. Evaporation can also lead to variability. To minimize evaporation, use PCR tubes/plates suitable for your thermocycler, make sure that the lids are not deformed, and minimize the time that the tubes are open while pipetting the ligation reaction by using a multichannel pipette.
Note that relative probe signals are not strongly influenced by the amount of DNA used within the recommended range, as long as this falls within the recommended range and as long as the DNA is free from impurities, as PCR primers are the limiting components of the PCR.
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