2.13 ELISA assay of inflammatory biomarkers
Levels of proinflammatory cytokines caused by RS-NC were measured using IL-6 and TNF-alpha ELISA assay. First, RS-NC (N/P ratio 5) containing 200 pmol of negative control siRNA were incubated for 30 minutes at RT. Then, 30 µL of RS-NC and DPBS as negative control were injected intradermally to three spots on epilated back of each five-week-old BALB/c mouse (Orientbio, Inc., Seongnam, South Korea). After three days, the skin tissue of euthanized mouse was excised, and skin samples were sectioned using a 5 mm biopsy punch (Kai Medical, Tokyo, Japan). Then, sectioned samples in lysis buffer (RayBiotech, Georgia, USA) were cut into small pieces using scissors (EpoS Medical, Tuttlingen, Germany) for one minute. Next, samples were homogenized using a homogenizer (T10BASIC, IKA, Staufen, Germany) at 8,000 rpm for 1 minute. ELISA assays were conducted using mouse IL-6 ELISA kit and mouse TNF-alpha ELISA kit (RayBiotech, Georgia, USA). Cytokines’ standard curves were obtained using four logistic parameters. Finally, cytokine levels were calculated by substituting absorbance values into the equation of a trend line.