It is possible to use the Promega Spectrum Compact or Hitachi Compact CE Sequencer DS3000 for MLPA. Coffalyser.Net™, our free MLPA® analysis software, offers initial support for both devices. However, some optimisation may be required to obtain green quality scores in your analyses.
Baseline correction
The Promega Spectrum Compact and Hitachi DS3000 devices do not perform a baseline correction on the raw data. As a result, the baseline is not expected to be around a signal of 0 RFU. In Coffalyser.Net, this can lead to increased baseline and baseline probes percentage cut warnings, even though the raised baseline is not necessarily a quality issue for these devices, as this is the expected behaviour.
This has been partially addressed by the application of an additional baseline correction step in Coffalyser.Net for the Promega Spectrum Compact device. At present, this baseline correction is not applied to the Hitachi DS3000 device, and also not to the size marker channels of either device.
Optimisation in Coffalyser.Net
To avoid unnecessary warnings related to the baseline in Coffalyser.Net, consider the following:
- If you use the Hitachi DS3000 and get one of the aforementioned baseline-related warnings for otherwise high-quality data, consider selecting the Promega Spectrum Compact device in Coffalyser.Net instead. This will apply the extra baseline correction step but will not alter any other settings, as the configuration of both devices is otherwise identical in Coffalyser.Net.
- We recommend using a size marker with a LIZ label for both devices. Size markers with a ROX label are less suitable, as these are more prone to baseline-related warnings for the size marker channel in Coffalyser.Net.
Electrophoresis
The (MS-)MLPA General Protocol contains instructions that can be used as a starting point for the electrophoresis settings on the Promega Spectrum Compact and Hitachi DS3000 devices. As stated above, we recommend using a LIZ-labelled size marker to avoid unnecessary warnings in Coffalyser.Net.