Total RNA and miRNA Isolation

On day 4 post transduction, the cells were washed with PBS once and detached mechanically. After spinning down (300 g for 5 min at 4°C) the biomaterial was briefly stored at −80°C until RNA was isolated. For total RNA extraction the RNeasy Plus Kit was used strictly according to manufacturer‘s protocol (Qiagen, Hilden, Germany). In brief, 350 μL of buffer RLT Plus were added to the collection tube containing the defrosted biomaterial and subsequently vortexed for 30 s. The transfer of the lysate to a gDNA eliminator spin column was followed by centrifugation for 30 s at 8,000 g. The flow-through was mixed with 350 μL of 70% ethanol (v/v) and transferred to an RNeasy spin column. After centrifugation for 15 s at 8,000 g, the column was washed by adding 700 μL of buffer RW1 to the column, followed by another centrifugation step for 15 s at 8,000 g. Thereafter, the column was washed twice with 500 μL of buffer RPE by centrifugation at 8,000 g, for 15 s and 2 min, respectively. After drying the membrane by centrifugation at full speed for 1 min, the column was placed into a new 1.5 mL collection tube. RNA was eluted by adding 30 μL of RNase-free water directly to the spin column membrane and centrifugation for 1 min at 8,000 g. For miRNA extraction, the miRNeasy and RNeasy MinElute Cleanup Kits were used according to the manufacturer‘s protocol (Qiagen) for enrichment of small RNAs from cultured cells. In brief, 700 μL QIAzol lysis reagent was added to the collection tube containing the defrosted biomaterial and homogenized by vortexing for 1 min. After incubation at room temperature for 5 min, 140 μL of chloroform were added and the lysate mixed by shaking the collection tube for 15 s, followed by incubation at room temperature for 3 min. Phase separation was accomplished by centrifugation for 15 min at 12,000 g at 4°C. 350 μL of the upper aqueous phase were transferred to a new reaction tube before and 525 μL of ethanol were added. After thorough mixing, 700 μL sample were transferred to an RNeasy mini column followed by centrifugation for 15 s at 8,000 g. This step was repeated with the remainder of the sample. The flow-through was transferred to a 2 mL collection tube and 500 μL of ethanol were added. Thereafter, the sample was transferred to an RNeasy MinElute spin column and centrifuged for 15 s at 8,000 g. This step was repeated with the remainder of the sample. The column was washed with 500 μL of buffer RPE by centrifugation for 15 s at 8,000 g and with 500 μL of 80% ethanol (v/v) by centrifugation for 2 min at 8,000 g. To dry the membrane, the column was centrifuged at full speed for 5 min. Then, the miRNA was eluted by adding 14 μL of RNase-free water to the spin column membrane followed by centrifugation for 1 min at full speed. The concentrations of the isolated miRNA and RNA of each condition were quantified using a NanoDrop spectrophotometer (Thermo Fisher). Thereafter, samples were stored at −80°C for further analysis.