2.3. miRNA Quantification by qRT-PCR

For miRNA quantification, an appropriate amount of Trizol LS (Thermo Fisher Scientific, Waltham, MA, USA) was used to prepare the samples for subsequent RNA extraction according to the manufacturer’s protocol. For miRNA validations, to account for the variation in RNA extraction efficiencies, 3 μL of a 5 μM synthetic miRNA oligonucleotide, cel-miR-39 (5′-UCACCGGGUGUAAAUCAGCUUG) (Integrated DNA Technologies, Leuven, Belgium), was added to each sample at the phenol extraction stage. In all cases, cDNA synthesis was performed by using the TaqMan MicroRNA Reverse Transcription Kit and the respective hairpin primers from TaqMan MicroRNA Assays (let-7a-5p assay ID:000377 and let-7b-5p assay ID:000378, miR-186-5p assay ID:002285; miR-210-3p assay ID: 000512; miR-16-5p assay ID:000391; miR-21-5p assay ID:000397 and miR-100-5p assay ID:000437; all from Thermo Fisher Scientific, Waltham, MA, USA). The quantification was performed by using the TaqMan Gene Expression Master Mix together with the respective TaqMan MicroRNA Assays on a Step-One Real-Time PCR instrument (Thermo Fisher Scientific, Waltham, MA, USA). shRNA detection was performed by synthesizing cDNA as above, but using custom-designed hairpin RT primers and amplified using KiCqStart SYBR Green qPCR Ready Mix (Sigma Aldrich, Saint-Louis, MO, USA) on a Step-One Real-Time PCR instrument, as above. The PCR efficiency of each reaction was calculated with the LinRegPCR program (developed by Dr. Jan Ruijter, Heart Failure Research Center, The Netherlands), while the Ct values were obtained from StepOne Software (Applied Biosystems, Waltham, MA, USA). In the case of overexpression studies, an equal amount of input RNA was used for reverse transcription. For miRNA validations, the total miRNA quantity was normalized to cel-miR-39 levels and further corrected for the volume of the respective fractions (Sepharose Fast Flow 4 fractionation volumes of 3–3.5 mL for EVs and 131–133 mL for non-EV (non-EV fraction + flow-through of 100 kDa ultrafiltration units)). For EV miRNA stability studies, EVs were isolated and purified as above, mixed with FBS at a final serum concentration of 10% and incubated at 37 °C for indicated times. At the end of each time point, Trizol LS was added to samples, and the RNA was extracted and analyzed by qPCR, as above.