ELISA to detect CHST15 siRNA

ELISA was performed to detect siRNA in the colon of normal mouse according to Yu’s method [26]. Colons were extracted at 4, 24, 48 and 144 hours after submucosal injection of CHST15 siRNA. In brief, total RNA of colon sample was transferred to 5% DMSO-PBS in a PCR tube and incubated at 80°C. The 3’-biotinylated template probe (Japan Bio Servies) solution was added to the sample solution and incubated. After hybridization, the sample solution was incubated in a streptavidin-coated 96-well plate. After washing, each well was incubated with the digoxigenin-labeled ligation probe (Japan Bio Servies) solution containing T4 DNA ligase (TaKaRa). Wells were washed with the washing buffer, with deionized water, and then incubated with the peroxidase-labeled anti-digoxigenin (1:1,000 dilution, Roche, Switzerland). After washing, enzymatic activity was detected using tetramethylbenzidine (TMB) (Sigma-Aldrich) and the absorbance was measured at 450 nm. To construct a standard curve, standards were prepared with known concentrations of CHST15 siRNA. The concentration of CHST15 siRNA in the colon was calculated from the standard curve.