We recommend confirming all abnormalities detected by MLPA or digitalMLPA whenever possible, using either a different (confirmation) probemix when available or an independent technique. Copy number changes detected by a single probe should always be confirmed.

It is essential to confirm copy number changes detected by a single probe. Single probes may more readily be affected by experimental issues or variability. In addition, a mutation or polymorphism in the probe's target sequence may lead to a reduced signal, which can even mimic a deletion. Sanger sequencing of the probe's target sequence can reveal if there are any variants in the probe's target sequence in such cases.

If available, you can use a specially designed confirmation probemix that has probes with different target sequences than the primary probemix. If a confirmation probemix is available, this is usually mentioned in the product documentation. It may also be possible to confirm results obtained with digitalMLPA with a conventional MLPA probemix, or vice versa. You can also use a variety of other techniques for confirmation purposes, including Sanger sequencing of (long-range) PCR products, qPCR, long-range PCR, NGS technologies, and array-CGH techniques. The method that is most suitable depends on the complexity of the region, the size of the aberration, and the availability of other techniques, among other things.