Cytotoxicity and optimization of siRNA transfection.

<p>HepG2 cells transfection experiment was optimized by treating the HepG2 cells with increasing amount of transfection reagent and concentrations of siRNA. (a) Result shows that the efficient silencing with minimal cytotoxicity was achieved using 1.0 µl of transfection reagent. (b) HepG2 cells transfected with increased concentration of GRP78 siRNAs and the result shows no evidence of toxicity regardless of the siRNA concentration as compared with scramble siRNA transfected HepG2 cells (One-way ANOVA with Dunnett's post-test, P>0.05). The optimal siRNA concentration is 50 nM. Similar results were observed in (c) CLTC siRNAs and (d) DNM2 siRNAs. (e) HepG2 cells were transfected and exposed to optimal siRNAs concentration (50 nM) that target GRP78, CLTC, and DNM2 in separated and combined transfection. Cytotoxicity was tested by measuring LDH level and results were confirmed by counting of viable cells using Trypan Blue exclusion assay. No evidence of cytotoxicity was observed for all pools of siRNA as well as in combined transfection (91.6%±2.0, 90.7%±1.9, 90.5%±0.4, and 89.9%±1.8 for GRP78, CLTC, DNM2, and combined transfection respectively). Trypan Blue exclusion assay showed also more than 90% viable cells. No significant difference was observed between separated and combined transfection by One-way ANOVA analysis (P>0.05). Results are expressed as mean ± SD from a representative experiment performed in triplicate.