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.