Every MLPA experiment should include at least three DNA reference samples and a no-DNA control. Reference samples should be derived from healthy individuals that are not expected to have any copy number changes or mutations in your region of interest. When using more than 21 samples, one additional reference sample should be added for every seven samples. You can also include positive controls (samples with a known deletion, duplication or point-mutation) if available, as these may aid in result interpretation.
MLPA is a relative technique based on the analysis of relative changes in probe signals. Thus, a given sample will not provide the information needed to estimate copy number changes without appropriate reference samples to compare to. Reference samples are samples where the target sequences of interest are assumed to have a ‘normal’ copy number. Usually, reference samples are DNA samples obtained from healthy individuals.
It is strongly recommended to use reference samples that have been purified using the same method and that are derived from the same type of tissue as the test samples. This minimizes non-biological differences between test and reference samples and minimizes structural variation. Multiple reference samples are needed to estimate each MLPA probe’s reproducibility within each experiment. In addition, this is also important to prevent false positive/negative results due to experimental issues or biological variation in the reference samples/reactions.
Reference samples in MLPA are required. An exception to this is when a large number (>20) of independent samples (from different families) are run at the same time and the chance for each individual probe being deleted or duplicated in a sample is low (<10%).
For more information, see our page on experimental setup.