In vitro screening and in vivo identification of rat IκBα-siRNA
10.3980/j.issn.1672-5123.2014.06.02
- VernacularTitle:大鼠IκBα基因siRNA序列的体外筛选及体内验证
- Author:
Rui, ZENG
;
Yu-Qing, LAN
;
Hai-Jun, GONG
;
Chi, ZHANG
;
Jin-Miao, LI
- Publication Type:Journal Article
- Keywords:
IκBα;
small interfering RNA;
rat ciliary muscles;
glaucoma;
uveoscleral outflow route
- From:
International Eye Science
2014;(6):986-991
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To seek a small interfering RNA ( siRNA ) sequence targeting rat inhibitor of nuclear factor kappa Bα ( IκBα) that can specifically and effectively suppress IκBα mRNA expression of rat ciliary muscles in vivo.METHODS:Three IκBα specific double stranded siRNAs were designed and synthesized. They were transfected into rat A7r5 cells which express IκBα gene. Flow cytometry was used to assess transfected efficiency. The mRNA and protein levels of IκBα were examined by Real Time quantitative polymerase chain reaction ( Real Time-PCR ) and western blot to screen a candidate valid sequence with the highest inhibitory rate. The Cy3 labeled non-specific control siRNA or the candidate valid siRNA was then injected into rat anterior chamber. Distribution of Cy3- siRNA in rat ciliary muscles was viewed by fluorescence microscopy, and the inhibitory effect in vivo of the valid siRNA was identified via Real Time-PCR and immunofluorescence. RESULTS: The suppression effect of the siRNA targeting the CTACGATGACTGTGTGTTT of IκBα gene was most obvious by vitro screening. By anterior chamber injection, this valid siRNA could reach rat ciliary muscles and effectively suppress IκBα gene expression with the highest inhibitory rate of 59. 0% on mRNA level at 24h after RNAi, and 52. 3% on protein level at 72h after RNAi (P<0. 01).CONCLUSION: It proves that the siRNA targeting the CTACGATGACTGTGTGTTT of IκBα gene is the valid sequence to suppress rat IκBα expression of ciliary muscles by RNAi in vivo.
