XMIR and AXMIR RNA Oligos

The XMIR and AXMIR kits allow for cell-mediated generation of ready to use exosomes packed with a miRNA (XMIR) or anti-miRNA (AXMIR) of choice. These exosomes can then be used to efficiently knock down native targets in recipient cells or be used to study biological pathways by which functional exosomal miRNA cargo is delivered to target cells. This technology is especially attractive as a means to study the efficacy of exosome mediated delivery of potential therapeutic miRNAs or anti-miRNAs to disease cells. To generate XMIRs and AXMIRS, an oligo is designed that fuses the XMotif sequence to a miRNA or anti-miRNA sequence, which results in exosomal loading of the RNA oligo. The oligo is first transfected into a culture of the exosome generating cell type of choice. IMPORTANT NOTE: Be sure to culture your exosome generating cell lines in media that does not contain standard FBS. There are high levels of cow exosomes present in FBS. Instead, use SBI's Exo-FBS Exosome-depleted FBS Media Supplement (cat# EXO-FBS-50A-1) in place of standard FBS media supplements. After 24 hours, exosomes packed with the XMIR or AXMIR are precipitated using ExoQuick-TC. After resuspension in PBS, the XMIR or AXMIR-loaded exosomes are ready to be added to target cells. Any miRNA, anti-miRNA or even siRNA sequence can also be used in a transfection-ready oligo for use in the XMIR/AXMIR system.

XMIR-1 and XMIR-122 packaging into exosomes

XMIR oligos for miR-1 and miR-122 were transfected at a 20 nM final concentration to test exosomal loading in HEK-293 cells. Transfection was accomplished using PureFection (catalog #LV750A-1), and exosomes were purified 24 hours later using ExoQuick-TC. RNA was extracted from exosomes, cDNA was synthesized, and qPCR was performed using the SeraMir kit (catalog #RA820A-1), Data were analyzed using an Applied Biosystems 7900HT qPCR instrument and relative exosome abundance levels were calculated using the delta-delta Ct method using exosomal miR-16 as a reference control.

Packaged XMIR-1 and XMIR-122 delivery to 3' UTR reporter cells

The XMIR-1 and XMIR-122 loaded exosomes were added to reporter HEK-293 cells previously transfected with a luciferase gene linked to the 3' UTR for MEF2A, a known miR-1 target, or RIMS1, a known miR-122 target, respectively. After 24 hours, luciferase assays were performed to determine bioactivity of the XMIR miRNAs delivered to target cells via exosomes. Notably, exosomes from cells transfected with 20 nM miR-1 oligo caused maximum luciferase down regulation, whereas exosomes from cells transfected with 100 nM miR-122 caused maximum luciferase down regulation, illustrating the need to optimize conditions for each XMIR/AXMIR oligo. The degree of knockdown XMIR-1 and XMIR-122 loaded exosomes displayed on the MEF2A and RIMS1 luciferase reporters are similar to that seen for transfections of miRNA oligos using a similar reporter assays, indicating that exosome mediated delivery of miRNAs occurs at maximal efficiency.