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Purpose   To seek for a small interfering RNA (siRNA) sequence targeting a cynomolgus monkey inhibitor of nuclear factor kappa B  α  (I κ B α ) that can specifically and effectively suppress I κ B α  gene expression of cynomolgus monkey ciliary muscle (CM) cells and trabecular meshwork (TM) cells in vitro and screen for optimal siRNA transfection concentration.Methods   Three I κ B α -specific double-stranded siRNAs were designed and synthesized. They were transfected into primarily cultured cynomolgus monkey CM cells and TM cells. The mRNA and protein levels of I κ B α  were examined by using real-time quantitative polymerase chain reaction (real-time PCR) and western blot to screen a pair of candidate valid sequences with the highest inhibitory rate. Both cells were transfected with Cy5-labeled nonspecific control-siRNA (NC-siRNA) of four different concentrations (10, 20, 50, and 100 nmol/L(nM)), and flow cytometry was used to assess transfection efficiency. Then, cells were transfected with the candidate valid I κ B α  -siRNA of the same four concentrations, and the cytotoxicity was detected by using Cell Counting Kit-8 (CCK8), and the inhibitory efficiency of I κ B α  was identified via real-time PCR to find out optimal siRNA transfection concentration.Results   The suppression effect of the siRNA targeting the GCACTTAGCCTCTATCCAT of I κ B α  gene was most obvious by in vitro screening. The inhibitory rate of I κ B α  was 82% for CM cells and 82% for TM cells on the mRNA level and 98% for CM cells and 93% for TM cells on the protein level, respectively. The results of flow cytometry showed that the transfection efficiency was the highest at 100 nM, which was 89.0% for CM cells and 48.2% for TM cells, respectively. The results of CCK8 showed that there was no statistically significant difference in cell viability after transfection of different concentrations of I κ B α -siRNA. The results of real-time PCR indicated that there was no statistical difference in the inhibitory efficiency of I κ B α  after transfection of different concentrations of I κ B α -siRNA.Conclusion   It proves that the siRNA targeting the GCACTTAGCCTCTATCCAT of I κ B α  gene is the valid sequence to suppress cynomolgus monkey I κ B α  expression of CM cells and TM cells by RNAi. 10 nM is the optimal transfection concentration.

In Vitro Screening and Transfection Concentration Optimization of Cynomolgus Monkey IκBα-siRNA