Genotyping
Genotyping was done using the GenoPharm® pharmacogenomics open array
panel which contains 120 assays for over 48 genes. GenoPharm® uses
TaqMan Assay chemistry using the Applied Biosystems QuantStudio 12K Flex
Real-Time PCR System (Thermo Fisher Scientific, Waltham, Massachusetts).
This open array system is customized for AiBST. In a 96 well plate, 6µl
of each 50 ng/µl of DNA sample was mixed with 6µl of the Open Array
Genotyping Master mix. The mixture was then shortly vortexed,
centrifuged and then transferred into a 384 well plate. A no template
control (NTC) was included on the plate. Using the robotic arm of the
Applied Biosystems Accufill System the contents of the 384 well plate
was transferred to an open array plate (33nl per well).The run was done
on the QuantStudio 12K Flex Real-Time PCR System using the default
settings pre-set by the manufacturer for an open array genotyping run.
All calls were made at cycle 40 using the default quality value ≥ 0.95
to assign a genotype call. Genotyping was done for UGT1A6 rs17863783,
SLC28A3 rs7853758 and RARG rs2229774 variants using a predesigned
primers and probes (Applied Biosystems). The thermal cycling conditions
was as follows; Initial denaturation 95ºC for 10 minutes, followed by 40
cycles of denaturation at 95ºC for 15 seconds and annealing and
extension at 60ºC for 60 seconds.