The optimal signal range for MLPA fragment peaks depends on the electrophoresis device. Care should be taken that signals fall between the minimum and maximum thresholds for each device, as otherwise this may lead to false results. Coffalyser.Net will issue a warning when a signal crosses a threshold, which results in quality penalties and renders the sample unsuitable for diagnostic analysis. When finding the optimal loading conditions of your capillary electrophoresis device, it is important to consider that some samples may have reduced or increased signals because of deletions or duplications, respectively.
Details
MLPA is a relative technique that involves comparison of relative peak heights to calculate a final ratio and to determine copy numbers of the target sequences of the probes (more details). This works because the amount of MLPA fragment for a probe changes depending on the copy number of its target sequence. For example, all else being equal, a probe targeting an autosomal sequence with a normal copy number of 2 will have 50% of the peak height of a normal sample in case of a heterozygous deletion (1 copy), and 150% of the peak height in case of a heterozygous duplication (3 copies). It is essential that all peaks have a signal in the detection range where this relationship holds. Signals that are too low may fall below the detection threshold; signals that are too high may start to flatten off.
The Coffalyser.Net Reference Manual lists the acceptable minimum and maximum signal height (in RFU) for all supported capillary electrophoresis devices. However, the optimal ranges are narrower, and Coffalyser.Net may show several warnings when signals start to approach the minimum and maximum values.
The table below lists RFU signal ranges within which Coffalyser.Net will not give warnings related to signal height.
| Supplier | Device(s) | Probe Signal Range (RFU)
Without Warnings* |
|---|---|---|
| Applied Biosystems | SeqStudio Genetic Analyzer | 375–20,800 |
| SeqStudio Flex Series Genetic Analyzer | 375–20,800 | |
| 3500 Series Genetic Analyzer | 375–20,800 | |
| 3730 Series Genetic Analyzer | 375–20,800 | |
| 3130 Series Genetic Analyzer | 375–4,000 | |
| PRISM 3100 Genetic Analyzer | 375–4,000 | |
| PRISM 310 Genetic Analyzer | 375–4,000 | |
| Hitachi | Compact CE Sequencer DS3000 | 375–20,800 |
| Promega | Spectrum Compact CE System | 375–20,800 |
| SCIEX/Beckman Coulter | GenomeLab GeXP | 3,750–120,000 |
| CEQ 8800 Genetic Analysis System | 3,750–120,000 | |
| CEQ 8000 Genetic Analysis System | 3,750–120,000 | |
| CEQ 2000 Genetic Analysis System | 3,750–120,000 | |
| Amersham | MegaBACE 1000 | 375–20,800 |
* Probes that target a sequence with a homozygous deletion have no signal and, therefore, do not cause warnings related to signal intensity.
When optimizing electrophoresis conditions, it is also important to keep in mind that samples with deletions and duplications (and thus lower and higher signals for some probes) still need to have all probes within the optimal range. In addition, not all probe signals are necessarily of equal height even in normal samples. Therefore, it is usually best to aim for peak heights around the middle of the ranges in the table above.