RNA extraction

Fresh-frozen samples were mechanically disrupted in 1 ml TRIzol Reagent (Life Technologies, Darmstadt, Germany) by using the TissueLyser II (Qiagen, Hilden, Germany) according to the manufacturers’ instructions. After the addition of 200 µl chloroform and a centrifugation step (14,000 × g, 10 min, 4 °C), the aqueous phase was collected and added to 250 µl isopropanol. Total RNA was extracted using the silica bead-based NucleoMagVet kit (Macherey & Nagel, Düren, Germany) with the KingFisher™ Flex Purification System (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturers’ instructions. In vitro-transcribed RNA of the enhanced green fluorescent protein (eGFP) gene was added during the extraction procedure as described by Hoffmann et al.⁵⁶.

RNA extraction from FFPE tissue was performed with a combination of truXTRAC FFPE total NA Kit (Covaris, Brighton, UK) and the Agencourt RNAdvance Tissue Kit (Beckman Coulter, Krefeld, Germany). FFPE sections of 3 µm thickness were loaded into a microTUBE-130 Screw-Cap (Covaris) together with 110 µl Tissue Lysis Buffer and 10 µl proteinase K solution (both Covaris). The lysate was processed with a M220 Focused ultrasonicator (Covaris) according to the manufacturer’s recommendations for acoustic pellet resuspension. The tube was subsequently incubated at 56 °C in a thermal shaker at 300 rpm overnight (no longer than 18 h). Subsequently, the sample tube was cooled to room temperature and centrifuged at 5000 × g for 15 min using a microTUBE-130 centrifuge adapter. A volume of 100 µl supernatant was transferred into a clean 1.5 ml reaction tube without transferring any wax or paraffin. After another centrifugation (5 min at 20,000 × g), 85 µl of the lower phase with the RNA-containing tissue pellet was transferred into a clean 1.5 ml reaction tube. It was incubated at 80 °C for 20 min and then cooled to room temperature, before 175 µl B1 buffer (Covaris) were added, mixed, and briefly centrifuged. Thereafter, 250 µl of 65% isopropanol were added, mixed, and briefly centrifuged. Subsequently, the preparations were further processed with the Agencourt RNAdvance Tissue Kit (Beckman Coulter) with the KingFisher™ Flex Purification System (Thermo Fisher Scientific) according to the manufacturer’s instructions.