Screening for siRNA sequence targetting rat Toll-like receptor 4 mRNA in vitro
10.3724/SP.J.1008.2008.00048
- Author:
Jin-Jun BIAN
1
Author Information
1. Department of Anesthesiology
- Publication Type:Journal Article
- Keywords:
Chemical synthesis;
PCR;
Small interfering RNA;
Toll like receptor 4
- From:
Academic Journal of Second Military Medical University
2010;29(1):48-52
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To screen for an optimized siRNA sequence targeting rat Toll-like receptor 4 (TLR4) in vitro. Methods: The full length gene of rat TLR4 was cloned and inserted into pEGFP-C1 plasmid to construct pEGFP-rTLR4. Three pairs of siRNAs targeting rTLR4 were chemically synthesized and were co-transfected with pEGFP-rTLR4 into HEK-293 cells via Lipofectamine2000. Cells were also co-transfected with siRNA targeting EGFP and negative control siRNA. The expression of EGFP was observed under inverted fluorescene microscope and flow cytometry. Results: Compared with the negative control group, 3 pairs of siRNAs targeting TLR4 and one pair of siRNA targeting EGFP significantly suppressed the EGFP expression (P<0.05); the inhibitory effect of siRNA2 (gene sequence: 5′-GTC TCA GAT ATC TAG ATC T-3′, 19 bp, 1 352-1 370) was the strongest one, with an interference efficiency over 75%. Conclusion: We have successfully obtained the siRNA sequence targeting TLR4 mRNA, which can efficiently suppress the expression of rat TLR4 mRNA in vitro.
