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, point-mutation or methylation status) 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 single 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 and, in the case of MS-MLPA, a ‘normal’ methylation status. 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 should be distributed “randomly” over the experiment to avoid bias. The image below shows an example.
Reference samples are required in every MLPA experiment. 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%).
When analysing tumour samples, reference samples should still be treated as similarly as possible. Ideally, reference samples are derived from similar, but healthy tissue, that has been treated in the same way as the test samples. This may include a FFPE-treatment (see this article for more details). For the detection of methylation changes in tumour samples, it is important to realize that methylation is cell-type specific, and can vary between tissues.
A no-DNA control is a reaction in which the 5 µl of sample DNA has been replaced with 5 µl TE0.1 (10 mM Tris-HCl pH 8.0 + 1 mM EDTA). Inclusion of a no-DNA control in every experiment is strongly recommended. The results can be used to check for contamination of reagents or equipment. More information on how to interpret a no-DNA reaction can be found in this article.
Positive control samples
Positive control samples can be used to check whether the entire MLPA procedure, including data analysis, is performed well. While positive samples can be useful, they are not required.
Like reference samples, positive control samples are preferably purified using the same method and from the same type of tissue as the test samples. If no positive control samples are available, suitable samples can sometimes be ordered from an online biorepository. MRC-Holland has very limited access to patient samples and cannot supply positive samples, but this article lists a large number of commercially available samples that have been tested with our products.