Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 45 In our pilot-study we used a faster, more efficient alternative for the subsequent MLPA analysis using the QIAxcel Advanced System. The proposed protocol enabled detection of exonic deletions/ duplications with the same resolution and accuracy as using a sequencer. The efficiency of the MLPA analysis using the QIAxcel Advanced System is more cost effective, compared to other detection systems in use. Materials and methods This pilot study was based on two different groups of clinical samples: • 12 FAP patients and 5 healthy controls • 9 HNPCC (hereditary non-polyposis colorectal cancer, Lynch syndrome) patients and 5 healthy controls MLPA The MLPA was performed using the SALSA® MLPA P043 APC probemix for the FAP samples and the SALSA MLPA P003 MLH1/MSH2 probemix (MRC-Holland® ) for the HNPCC samples following the manufacturer’s manual. Amplicon visualization The separation and visualization of MLPA-generated amplification products was performed using both a sequencer and QIAxcel Advanced System (QIAGEN) for capillary electrophoresis, following the manufacturer’s instructions for the sequencer and a modified protocol for the QIAxcel Advanced System (QIAGEN). Samples were separated using the QX DNA High Resolution Kit with a customized 0M1100_2kV_AM10s_S5kV method, with the following electrophoresis parameters: alignment marker injection at 4 kV for 10 s, sample injection at 5 kV for 10 s and separation at 2 kV for 1100 s. Alignment marker 15 bp/600 bp was run simultaneously with the samples and the size was estimated in comparison with the QX DNA size marker 25 bp–500 bp. Data analysis Analysis of the data generated by the sequencer was performed with Coffalyser® software (MRC-Holland), while QIAxcel ScreenGel® 1.2 software (QIAGEN) was used for data analyses of the results from the QIAxcel Advanced System. Note: The layouts of all graphics in this Application Note are slightly changed from originals to more clearly visualize the results. No modifications were conducted that influenced the results themselves and the authors will provide original graphics/data on request.

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