Saliva-based genotyping protocol

Genomic DNA extracted from saliva was used for genotyping analysis. Briefly, for saliva collection, saline mouth solution to rinse in the mouth for 60 s was used. Therefore, the genomic DNA was extracted from buccal epithelial cells from saliva samples as previously described [23 (link)]. Quantification of the concentration and purity of the DNA was determined by spectrophotometry (Nanodrop 1000; Thermo Scientific, Wilmington, DE, USA).
Six SNPs were evaluated in the present study and are reported in Table 1. The genotyping was blindly performed using the Taqman™ method for real-time PCR in the StepOnePlusTM sequence detection system, Applied Biosystems™ (Foster City, CA, USA) or in the Mastercycler® ep realplex-S thermocycler, Eppendorf AG (Hamburg, Germany). Additionally, 10% of the sample was genotyped twice and an agreement of 100% was observed. The reaction was previously described in [24 (link)].