So131

RNA isolation was performed as previously described according to MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines [17 (link), 30 (link)]. A total of 500 µl peqGOLD TriFast^TM (PEQLAB Biotechnology GmbH, Erlangen, Germany) was added per well and further processed according to the manufacturer’s instructions. The resulting RNA pellet was resuspended in 20 µl nuclease-free doubly distilled water (H₂O_dd; T143, Carl Roth, Karlsruhe, Germany). RNA was quantified using a NanoPhotometer (N60; Implen, Munich, Germany). A total of 100 ng RNA per sample was transcribed into cDNA using 1 µl oligo-dT18 primer (SO131, Thermo Fisher Scientific Inc., Waltham, MA, USA), 1 µl random hexamer primer (SO142, Thermo Fisher Scientific Inc.), 1 µl dNTP mix (L785.2, Roti®-Mix PCR3, Carl Roth), 1 µl RNase inhibitor (EO0381, Thermo Fisher Scientific Inc.), 1 µl MLV-reverse transcriptase (M1705, Promega, Fitchburg, WI, USA), 4 µl 5 × M-MLV-buffer (M1705, Promega) in a total volume of 20 µl by addition of nuclease-free H₂O_dd (T143, Carl Roth). Samples were incubated for 60 min at 37 °C and 2 min at 95 °C. Reverse transcription was performed for all samples at the same time to minimize experimental variation.

Total RNA (500 ng) were reverse transcribed with random primers (48190–011, Thermo Fisher Scientific), oligo (dT) (SO131, Thermo Fisher Scientific), and deoxynucleotide (dNTP, 10297–018, Thermo Fisher Scientific) using Superscript® III reverse transcriptase (18080–044, Thermo Fisher Scientific). Thermocycling conditions were 10 min, 25°C; 60 min, 55°C; and 15 min, 75°C.

For gene expression analysis, RNA was isolated from cell pellets using 0.5 mL peqGOLD TriFast™ (072319-30, PEQLAB, Erlangen, Germany) per well, and processed according to the manufacturer’s instructions. The resulting RNA pellet was reconstituted in nuclease-free water (T143, Carl Roth, Karlsruhe, Germany), and after photometrical adsorption measurements (280 nm and 260 nm; NanoDrop, Implen, Munic, Germany), cDNA synthesis was done according to a previous published protocol [25 (link)]. Therefore, 0.1 µg RNA was substituted with 2 µL 5x M-MLV-buffer (M1701, Promega, Madison, WI, USA), 0.5 µL dNTP mix (L785.1/2, Carl-Roth, Karlsruhe, Germany), 0.5 µL RNase inhibitor (40U; EO0381, Thermo Fisher Scientific, Waltham, MA, USA), 0.5 µL oligo-dT18 primer (0.1nmol; SO131, Thermo Fisher Scientific, Waltham, MA, USA), 0.5 µL M-MLV reverse transcriptase (M170B, Promega, Madison, WI, USA) and replenished to 10 µL total volume with nuclease-free water (T143, Carl-Roth, Karlsruhe, Germany). After 60 min incubation at 37 °C and 2 min at 95 °C, samples were diluted to a final concentration of 1 ng/µL.