Detection of immune response activation by exogenous nucleic acids by a multiplex RT-PCR method

Transfection of mammalian cells or in vivo administration of nucleic acids can induce inflammatory cytokines and/or interferon response, which could significantly influence the ex vivo or in vivo applications of gene-targeting strategies based on nucleic acids. Further induction of the interferon and inflammatory related stress responses may result in off-target effects and toxicity. This work describes an original one-step multiplex reverse transcription polymerase chain reaction procedure, which allows testing the induction of interferon and proinflamatory related responses by nucleic acids in the cell system of choice. The developed procedure has been tested on mammalian cells transfected with ssRNA, dsRNA, enzymatically synthesized siRNA and synthetic oligodesoxyribonucleotides containing unmethylated cytosine-guanosine motifs. This procedure is a rapid and convenient screening assay that could be used routinely in both the clinical and the research laboratory to validate the stimulation of the immune system on mammalian cells by nucleic acids.