Lightcycler 480 endpoint genotyping software

10 to 20 ng of DNA of each patient were used for TaqMan SNP genotyping assays (comprising primers and fluorescent probes) according to the manufacturer’s instructions (Thermofisher Scientific, Waltham, MA USA). Genotyping was performed by real–time PCR using either allelic discrimination in the 7300 RT–PCR System (Thermofisher Scientific, Waltham, MA USA), either using endpoint genotyping in an LightCycler 480II (Roche Molecular Diagnostics Pleasanton, CA, USA). For the 7300 RT–PCR System PCR parameters involved an initial denaturation at 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Post run data were analysed by 7300 SDS software (Thermofisher Scientific, Waltham, MA USA) and Automatic calls were assigned with approximately 99.8% quality. A call rate > 95% was considered the cutoff to consider genotyping. For LightCycler 480II PCR parameters involved an initial denaturation at 95 °C for 10 min followed by 40 cycles at 95 °C for 10 s, 60 °C for 1 min, and 72 °C for 10 s. Post run data were analysed by LightCycler 480 Endpoint Genotyping software (Roche Molecular Diagnostics Pleasanton, CA, USA). Two blank (water) controls in each 96-well plate were used for the assay quality control.

We will genotype several SNPs in ADRB2, OPRM1, COMT, UGT2B7, and IL-1Ra genes in all patients by the Taqman SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA) in the Pain-OMICS core-lab of Azienda Ospedaliera of Parma.
The peripheral blood samples are collected during general anesthesia into EDTA-coated tubes. We extract DNA by using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) from 200 μl of blood. DNA obtained are stored at −20 °C for subsequent TaqMan analysis. Amplification is performed in a volume of 5 μl containing 10 ng of genomic DNA, SsoFast Probes Supermix (Bio-Rad Laboratories S.r.l, Segrate, Italy). Cycling and hybridization conditions are set in accordance with the instructions of the manufacturer.
The amplification and genotyping discrimination are performed in a LightCycler 480 Real-Time PCR System (Roche Diagnostics Ltd, Lewes, UK). All assays are used in accordance with the instructions of the manufacturer (Life Technologies, Monza, Italy). The SNP calling is made with LightCycler 480 Endpoint Genotyping software version 1.5.0.39 (Roche Diagnostics Ltd).