Quantstudio three cycler

The miRNA amount of the EV samples was calculated with the Qubit™ microRNA Assay Kit (Thermo Fisher Scientific, Germany). cDNA was synthesized using TaqMan^® MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Germany). RT-PCR was performed using TaqMan^® universal PCR Master Mix (Thermo Fisher Scientific, Germany) on a Quantstudio three cycler (Applied Biosystems, Germany). The following miRNAs were examined: let7a, miR-10a, miR-21, miR-23, miR-155, miR-221, miR-222 and miR-486, miR29a, miR34a. All primers were purchased from Applied Biosystems (Supplementary Table S2). The samples were run in duplicates. U18 was used as endogenous control according to Vriens et al. (Vriens et al., 2012 (link)). Amplicons were normalized to U18 applying the comparative CT (∆CT) method.

Total RNA were isolated from CD34⁺ HSPCs after incubation with EVs using RNeasy Micro Kit (Qiagen, Germany) and reverse transcribed into cDNA using RevertAid cDNA synthesis kit (Thermo Fisher Scientific, Germany) with oligo-dT primers. Relative target quantity was determined using the comparative CT (∆∆CT) method. RT-PCR was performed using SYBR Green/ROX PCR master mix (Thermo Fisher Scientific, Germany) and target specific primers for PTEN, CDKN2A and SIRT1 (Supplementary Table S4) on a Quantstudio three cycler (Applied Biosystems, Germany). Amplicons were normalized to endogenous GAPDH control. All samples were run in duplicates.