miRNA expression analysis using NanoString nCounter technology

For miRNA expression analysis the NanoString human v3 miRNA panel (NanoString Technologies), targeting 799 miRNAs, was used according to the manufacturer’s instructions. Briefly, 100 ng of total RNA was used with 20 hours hybridization. Subsequently, the hybridization reaction was analyzed using the nCounter™ SPRINT (NanoString Technologies) platform and the raw data were processed using the nSOLVER 3.0 software; first, a background subtraction of the geometric mean of the negative controls and a positive control normalization using the geometric mean of all positive controls was performed. A second normalization followed using the geometric mean of the housekeeping genes RPL19 and RPL0. In addition, a sample calibration was performed for batch effect correction introduced by using code sets with different LOT numbers.