Korean J Anesthesiol.  2010 Dec;59(6):403-410. 10.4097/kjae.2010.59.6.403.

The optimal concentration of siRNA for gene silencing in primary cultured astrocytes and microglial cells of rats

• Ki KH
• Park DY
• Lee SH
• Kim NY
• Choi BM
• Noh GJ

• KMID: 1766501
• DOI: http://doi.org/10.4097/kjae.2010.59.6.403

Abstract

BACKGROUND
Small interfering RNAs (siRNAs) have been used to knockdown specific gene expression in various cells. Astrocytes and microglial cells play a key role in fundamental central nervous system functions and in chronic neuroinflammation. The aims of this study were to determine the optimal concentration of siRNA demonstrating efficient transfection and inhibition of gene expression via RNA interference (RNAi) and lower cytotoxicity, in primary cultured astrocytes and microglial cells of rats.
METHODS
Astrocytes and microglial cells were isolated from the cerebral cortices of 2-day-old rats. Both the cells were transfected using transfection reagent (Lipofectaminetrade mark 2000), and fluorescein-labeled double-stranded RNA (dsRNA) or siRNA targeting green fluorescent protein. Transfection efficiency and cytotoxicity of dsRNA, and the degrees of RNAi induced by siRNA in these cells, were evaluated at various concentrations of RNA.
RESULTS
Transfection efficiencies of dsRNA in both astrocytes and microglial cells were significantly higher (P < 0.05) at the concentrations of 20, 40, and 80 nM than at the concentrations of 0, 5, and 10 nM. There were no significant cytotoxicities within the applied concentrations of dsRNA (0-80 nM). The degrees of RNAi induced by siRNA were significantly higher (P < 0.05) at the concentrations of 5, 10, 20, 40, 80 nM, and 20, 40, 80 nM in astrocytes and microglial cells, respectively, compared with the control (0 nM).
CONCLUSIONS
The siRNA concentration of 20 nM may be appropriate to induce RNAi in both astrocytes and microglial cells, while demonstrating low cytotoxicity, high transfection efficiency, and effective RNAi.

Keyword

Cytotoxicity; RNA interference; Small interfering RNA; Transfection efficiency

MeSH Terms

Animals
Astrocytes
Central Nervous System
Cerebral Cortex
Gene Expression
Gene Silencing
Rats
RNA Interference
RNA, Double-Stranded
RNA, Small Interfering
Transfection
RNA, Double-Stranded
RNA, Small Interfering