Taqman probe based assays

Polymorphisms in EGFR, ABCG2, ABCB1, and POR (Online Resource, Appendix A) were analyzed using TaqMan® probe-based assays (Applied Biosystems, Foster City, CA, USA), whereas the ABCG2 polymorphism (rs2231137) was studied using the CycleavePCR® assay (TaKaRa Bio Inc., Kusatsu, Japan). The detailed genotyping method is provided in the Online Resource (Appendix A).

Saliva samples were collected from all participants using the Oragene DNA collection kit and DNA was extracted (DNA Genotek, Ontario, Canada). Three polymorphisms in the TAS2R38 receptor gene (rs1726866, rs10246939 and rs713598) define the genotype. The first two were genotyped with the Omni Express 700k Illumina Chip. The third one was analysed using TaqMan probe-based assays (Applied Biosystems, Foster City, CA, USA).

The expression of selected transcripts was evaluated by retrotranscription and quantitative PCR (RT-qPCR) by using TaqMan probe-based assays (Applied Biosystems, Foster City, CA, USA). Total RNA extraction and retrotranscription were performed as described in the Microarray processing section. PCR was performed in a 20 μL total volume reaction by mixing 100 ng cDNA, 2X TaqMan Universal Master Mix II with UNG (Applied Biosystems, Foster City, CA, USA), 1μL of specific target TaqMan assay probe, and 1 μL of primer limited probe for the endogenous control glyceraldehyde-3-phosphate deshydrogenase (GAPDH). Amplification was conducted at 50 °C for 2 min, 95 °C for 10 min as initial step followed by 95 °C for 15 sec, 60 °C for 1min for 40 cycles on a StepOne Real Time PCR System (Applied Byosistems, Foster City, CA, USA). Relative gene expression was calculated using the 2^ΔΔCT method.