Amicon ultra centrifugation tubes

The negative control miRNA (cat. no. SMC-2003), miR-21 mimic (5′-UAGCUUAUCAGACUGAUGUUGA-3′; cat. no. SMM-002) and miR-21 inhibitor (5′-AUCGAAUAGUCUGACUACAACU-3′; cat. no. SMI-002) were obtained from Bioneer Corporation (Daejoen, Korea). The miRNAs were transfected into human peripheral blood-derived exosomes using the Exo-Fect™ Exosome Transfection kit (System Biosciences) according to the manufacturer's protocol. Next, the exosomes were washed twice using Amicon ultra centrifugation tubes (EMD Millipore, Billerica, MA, USA) with cold PBS. The transfected exosomes were directly used for further experiments.

The NC-siRNA and siRNA targeting rat RAGE (sense, 5′-CUC UAC GAU CCC AAU UCA A dTdT-3′; antisense, 5′-UUG AAU UGG GAU CGU AGA G dTdG-3′)³⁸ (link) were obtained from Bioneer (Daejeon, Korea) and transfected into sEVs using Exo-Fect (System Biosciences, Mountain View, CA, USA) in accordance with the manufacturer’s instructions. Thereafter, sEVs were incubated with RNase A (Sigma) and washed twice with cold PBS using Amicon ultra centrifugation tubes (EMD Millipore, Billerica, MA, USA). siRNA retention in sEVs was assessed by measuring fluorescence at a maximum excitation emission spectrum of 493–517 nm on a FlexStation 3 fluorescence microplate reader (Molecular Devices). Fluorescence in each sample was compared with the input of FAM-NC-siRNA alone.