Rn_LOC311846_1 FlexiTube siRNA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	After four hours of incubation, add serum to cells. After 24 hours replace Opti-MEM with DMEM and repeat the same process thrice to increase the transfection efficiency.	Quantify after 48 h post transfection.

Protocol tips
After four hours of incubation, add serum to cells. After 24 hours replace Opti-MEM with DMEM and repeat the same process thrice to increase the transfection efficiency.
Downstream tips
Quantify after 48 h post transfection.

Publication protocol

Gene expression knockdowns were performed using commercially available gene-specific siRNA constructs listed in Table​Table1.1. Cells grown to ∼50–70% confluency were transfected with one or several siRNAs and Lipofectamine® RNAiMAX reagent as previously optimized for primary astrocytes (Bowens et al. 2013). As a negative control we used Alexa-488-labelled Allstars scrambled siRNA from Qiagen. After 4–8 h of incubation with siRNA/Lipofectamine complexes, transfection media were replaced with fresh culture media. This transfection technique produces negligible cell toxicity. Changes in the mRNA expression levels were quantified at 48 h post-transfection, and all functional assays were performed at 72 h post-transfection. Based on the Alexa-488 fluorescence analysis in negative controls, the efficacy of transfections routinely reached 95%.

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Manufacturer protocol

Download the product protocol from Qiagen for Rn_LOC311846_1 FlexiTube siRNA below.

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