Polytron homogenizer pt3100

Snap frozen skin biopsies were thawed in wet ice and homogenized in a Polytron Homogenizer PT3100 (Kinematica AG, Switzerland) with TRIzol Reagent. RNA was isolated following the TRIzol Reagent standard manufacturer’s protocol (Ambion, Thermo Fisher Sci., MA, USA). Contaminating DNA was removed using the DNA-free kit according to the manufacturer’s protocol (Thermo Fisher Scientific Inc., MA, USA). RNA integrity was assessed in 1% agarose gel electrophoresis and TapeStation RNA ScreenTape (Agilent Technology, CA, USA).

Snap frozen skin biopsies were thawed in wet ice and processed using TRIzol Reagent (Ambion, Thermo Fisher Scientific Inc., MA, USA) according to the manufacturer’s instructions with the help of Polytron Homogenizer PT3100 (Kinematica AG, Switzerland). RNA was treated with DNAse using the DNAfree kit (Thermo Fisher Scientific Inc., MA, USA) according to the standard manufacturer’s protocol, prior to use for library preparation and RT-qPCR. RNA integrity was assessed in 1% agarose gel electrophoresis or TapeStation RNA ScreenTape (Agilent Technology, CA, USA). During RNA isolation, samples were randomly assigned to extraction batches and freeze-thaw cycles to minimize batch effects and the introduction of technical artifacts. All procedures applied to samples were carried out using reagents from the same lot. The first author conducted the experiments aware of each sample group during the entire process, therefore, no blinding scheme was used, although we do not rely on perceptual/abstract measurements or analyses nor did we purposefully exclude samples.