In Vitro Screening and Transfection Concentration Optimization of Cynomolgus Monkey IκBα-siRNA. (15th April 2020)

• Record Type: Journal Article
• Authors: Ou, Zhaoxing; Zeng, Rui; Lin, Yifan; Zhang, Si; Alzogool, Mohammad; Zeng, Peng; Lan, Yuqing
• Other Names: Zarnowski Tomasz Academic Editor.
• Is Part Of: Journal of ophthalmology. Volume 2020(2020)
• Publication Date: 2020-04-15
• Subjects: Ophthalmology -- Periodicals; Eye Diseases; Ophthalmology; Ophthalmology; Electronic journals; Periodicals; Periodicals; Fulltext; Internet Resources; Periodicals; 617.7
• DOI: 10.1155/2020/1848540
• Languages: English
• ISSNs: 2090-004X

Abstract

Abstract : 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.