Digital PCR (dPCR)
For the allele-specific expression assay, a custom TaqMan dPCR was
carried out taking advantage of the QuantStudio 3D Digital PCR System
(Applied Biosystems) and according to the manufacturer’s
recommendations. Specific TaqMan probes were designed with the Custom
TaqMan Assay Design Tool (Thermo Fisher Scientific) in order to
recognize the mutant (FAM) or the wild-type (VIC) allele. The dPCR was
used to analyze tumor cDNA from the carriers; cDNA from sporadic CRC and
healthy colon tissue were used as controls.