Peqstar 96 universal gradient

Quantitative real time PCR (qRT-PCR) was used to validate the miRNA expression profile of the selected miRNAs in an independent sample set. The RNA was isolated from ARPE-19 cells by miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. 100–300 ng of RNA were retro transcribed using TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) using specific TaqMan RT primers and the thermocycler PeqSTAR 96 Universal Gradient (PeqLAB, Erlangen, Germany), the cycles used were 16 °C/30 min, 42 °C/30 min, 85 °C/5 min and 4 °C/infinity. Quantitative real time PCR was performed using TaqMan™ microRNA Assays (Thermo Fisher Scientific, Waltham, MA, USA) with TaqMan Gene Expression master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and RT-PCR Roche 234 LighterCycler 480 with the appropriate temperature cycles (50 °C/2 min, 95 °C/10 min, 40 cycles: 95 °C/15 s and 60 °C/1 min). Normalisation was performed with RNU6B snoRNA and RNU43 snoRNA. Relative expression was calculated as 2^−ΔΔCt.

To perform microarray analysis, ARPE-19 cells from 4 separate cultures were exposed to control and H₂O₂ 600 µM treatment for 24 h. Total RNA was extracted using SeraMir Kit (System Biosciences, Mountain View, CA, USA) according to manufacturer’s instructions. Therefore, four microarrays were performed for each condition: control, ARPE-19 cells exposed to H₂O₂ 600 µM, sEVs released by control cells and sEVs released by ARPE-19 cells exposed to H₂O₂ 600 µM.
Total RNA quantity and quality (260/280 absorbance ratio) were assessed using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA was reversely transcribed (cDNA synthesis) using PeqSTAR 96 Universal Gradient (PeqLAb, Erlangen, Germany) under the following conditions: 60 °C/5 min, RT/2 min, 42 °C/30 min, 95 °C/10 min and 15 °C/hold. Real-time quantitative PCR (qPCR) was performed using 384 well SeraMir Profiler using RT-PCR QuantStudio^TM 3 y 4 system (Thermo Fisher Scientific, Waltham, MA, USA) with the appropriate temperature cycles 50 °C/2 min, 95 °C/10 min, 40 ciclos; 95 °C/15 s, 6 °C/1 min. The miRNA expression values were calculated using three endogenous controls: RNU43, RNU1Q and RNU6. The expression was calculated according to the 2^−∆∆Ct method.