Immunological off-target effects of RNA and RNAi therapy are a considerable challenge in research and the future of RNA-therapy. Here we investigated some of the hurdles we previously encountered when transfecting mircroRNA (miRNA) control sequences into chondrocytes in an inflammatory model simulating osteoarthritis (OA). We investigated different negative control sequences of different technologies; Pre-miR miRNA Precursor and mirVana from Thermo Fisher Scientific. We used RT-qPCR, western blot analysis and mass spectrometry to asses for the effects of the transfected control sequences.
The data did not show a global immunological off-target effect, however a specific off-target effect on IL6 and IL8 was observed. IL6 and IL8 were both upregulated by the negative control from the Pre-miR miRNA Precursor technology (Pre-neg #1), and downregulated by the negative control from the mirVana technology (mirVana-neg). Moreover, the results suggested that the effect on IL6 and IL8 was dependent on both sequence and type of chemical modifications in addition to donor variation. We conclude that negative controls should be selected wisely, and suggest that scientists need to test several controls to ensure correct interpretation of data before drawing any conclusions.