Droplet digital PCR

The droplet digitalPCR (ddPCR) was applied to DNA extracted by exosome (exoDNA) samples isolated from plasma to evaluate 37 WT KRAS patients and 33 mutant KRAS patients. ddPCR probes matching the G12V e G12D KRAS mutations were purchased from Bio-Rad. Droplet digital PCR allow to distinguish the mutation forms thanks to ddPCR probes (FAM probes for KRAS G12V or G12D gene mutation and HEX probe for WT gene) matching the G12V e G12D KRAS mutations and the WT gene in the same sample. We adopted the ddPCR KRAS G12/G13 Screening Kit to screen our KRAS WT patients (we could not know what mutation had occurred at progression step) for the following seven KRAS mutations in a single well: G12A, G12C, G12D, G12R, G12S, G12V, G13D.

ddPCR was carried out on a QX100ddPCR system (Bio-Rad Laboratories). A total volume of 22 µl PCR reaction mixtures was prepared with 10 µl of Supermix for probes without dUTP (Bio-Rad), 1 µl target primers/probe, and DNA sample/water (variable volume). The DNA template input volume used for the analysis was 10μL. Using the QX100 Droplet Generator (Bio-Rad) according to the manufacturer’s instructions, a mean of 14,000 droplets per sample were obtained from the PCR reaction. Samples were then transferred to a Bio-RadQX-100 droplet reader and analyzed based on fluorescence intensity by QuantaSoft v1.4.0.99 software from Bio-Rad. The DNA concentrations were estimated by the Poisson distribution. Fractional abundance (FA) was calculated as follows: FA (%) = [mutant copy/ (wild-type + mutant copy)] × 100.