*2.5. RT-qPCR*

Knockdown efficiencies on the mRNA level were analyzed by RT-qPCR. Seventy-two hours after transfection, the total RNA was isolated using a combined TRI reagent (TRIfast, VWR, Darmstadt, Germany) and silica column protocol as previously described [52]. One microgram of total RNA was reverse transcribed using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific; Schwerte, Germany). The cDNA synthesis mixture was incubated at 25 ◦C for 10 min, 42 ◦C for 60 min, and heat denatured at 70 ◦C for 10 min.

For quantitative real time PCR, the cDNA was diluted 1:10 with diethyl pyrocarbonate (DEPC)-water and 4 μL were mixed with 5 μL PerfeCTa SYBR Green FastMix (Quantabio, Beverly, MA) and 1 μL 5 μM primer mix. The RT-qPCR was performed using a Real-Time PCR System (Applied Biosystems) with the following instrument settings: pre-incubation at 95 ◦C for 2 min, followed by 45

amplification cycles (95 ◦C for 15 s, 55 ◦C for 15 s, 72 ◦C for 15 s). Levels were normalized for RPLP0. Primer sequences are given in Table S2.
