Digital PCR

Quantification of allele-specific mRNA expression was performed using the QuantStudio 3D Digital PCR System (Thermo Fisher Scientific). SNP⁴⁹ (rs45441892) was detected by the human TaqMan SNP Genotyping Assay C_27850890_10 and SNP¹⁵⁶ (rs45571738) by the human TaqMan SNP Genotyping Assay C_27837236_10, respectively. Each reaction mix for one QuantStudio 3D Digital PCR chip v2 consisted of QuantStudio 3D Digital PCR Master Mix v2 (Thermo Fisher Scientific), specific human TaqMan SNP Genotyping Assay (primer/probe mix), template cDNA or gDNA with final concentration at 3.45 ng/µl and PCR-grade water in a final volume of 14.5 µl. PCR conditions were as follows: stage 1 enzyme activation, 96°C for 10 min; stage 2 annealing/extension and new denaturation at 54°C (SNP⁴⁹) or 58°C (SNP¹⁵⁶) for 2 min and 98°C for 30 s (39 cycles); stage 3 annealing/extension 60°C for 2 min and storage at 10°C. Each sample was analyzed using a separate 20,000 times compartmented QuantStudio 3D Digital PCR chip v2 and the AnalysisSuite Software (Thermo Fisher Scientific). The expressed allele amounts were presented as copy numbers per microliter PCR mixture.